We thank Stephan Wilkens for help in transmission electron microscopy. This work was supported by the National Institutes of Health Grant R01AG023731.

Previous studies have shown that the first amino-terminal 22 residues of Mgm101 are cleaved after import into mitochondria 19. For expression in Escherichia coli, the MGM101 open reading frame lacking the first 22 codons is amplified by PCR and placed downstream of the malE sequence encoding the maltose binding protein (MBP) in a modified version of the expression vector pMAL-c2E. This generates the MBP-Mgm101 fusion with a linker containing a cleavage site for the PreScission protease (Figure 3). The plasmid is first constructed by selecting E. coli transformants without the IPTG and Xgal blue/white selection. The resulting plasmid pMAL-c2E-MGM101 is then introduced into the E. coli strain BL21-CodonPlus(DE3)-RIL by selecting ampicillin and chloramphenicol resistant colonies.
1. Expression, Induction, Cell Lysis and DNase I Treatment
<ol>
	<li>Inoculate fresh transformants in 10 ml of LB medium (1% Bacto tryptone, 0.5% yeast extract, 1% NaCl) supplemented with glucose (0.2%), ampicillin (100 &mu;g/ml) and chloramphenicol (50 &mu;g/ml). Incubate at 37 &deg;C O/N with shaking at 200 rpm.</li>
	<li>Inoculate the 10 ml preculture into 2 liters of the supplemented LB medium as above. Grow the cells at 37 &deg;C with shaking until OD600 reaches 0.5.</li>
	<li>Induce expression of the MBP-Mgm101 fusion protein by adding Isopropyl &beta;-D-1-thiogalactopyranoside (IPTG) at a final concentration of 0.3 mM. Grow the cells with shaking at 30 &deg;C for 5 hr.</li>
	<li>Collect the cells by centrifugation using a Beckman JA-10 rotor (5,500 x g, 4 &deg;C, 10 min). After discarding the supernatant, resuspend the cell pellet in 30 ml of lysis buffer (20 mM Tris-HCl, pH 7.4 and 1 mM EDTA, pH 7.4) containing protease inhibitors (25 &mu;M leupeptin, 1 &mu;M pepstatin A and 1 mM PMSF).</li>
	<li>Sonicate cell suspension on ice for 20 sec using an ultrasonic processor (Heat Systems; Model W-385) at 50% duty cycle, allow to cool on ice for 30 sec, and repeat 2x.</li>
	<li>Add 1 ml of DNAse 1 stock at 2 mg/ml. Rock the cell lysate at 4 &deg;C for 2 hr.</li>
	<li>Adjust NaCl to a final concentration of 500 mM.</li>
	<li>Remove the cell debris by centrifugation at 10,000 x g at 4 &deg;C for 30 min.</li>
</ol>
2. Purification with Amylose Affinity Chromatography
<ol>
	<li>Equilibrate 1.5 ml of amylose resin (50% slurry) in the lysis buffer. Add the equilibrated amylose resin to the cell lysate. Rock gently at 4 &deg;C O/N.</li>
	<li>Load the lysate-resin mix onto an Econo-Column chromatography column installed in a cold room. Allow the unbound proteins to pass the column by gravity.</li>
	<li>Wash the amylose resin with 300 ml of the wash buffer I (20 mM Tris-HCl, pH 7.4; 400 mM NaCl; 1 mM EDTA, pH 7.4; and 0.2 mM PMSF).</li>
	<li>Repeat the wash with 150 ml of wash buffer II (20 mM Tris-HCl, pH 7.4; 200 mM NaCl, 1 mM EDTA, pH 7.4; and 0.2 mM PMSF).</li>
	<li>Add 5 ml of elution buffer (20 mM Tris-HCl, pH 7.4; 200 mM NaCl; 1 mM EDTA, pH 7.4; and 0.2 mM PMSF, 10 mM maltose) to the column, incubate at 4 &deg;C for 10 min, collect the eluate and repeat for more times.</li>
	<li>Combine the eluates, determine the yield and purity of MBP-Mgm101 by loading an aliquot of the eluate on a SDS-PAGE gel followed by coomassie staining (Figure 4).</li>
</ol>
3. PreScission Protease Cleavage and Cation Exchange Chromatography
<ol>
	<li>Add 50 units of PreScission protease to the MBP-Mgm101 eluate. Perform the cleavage at 4 &deg;C O/N and allow it to continue during dialysis against the dialysis buffer (20 mM Tris-HCl, pH 7.4; 100 mM NaCl; 2 mM DTT; 1 mM EDTA, pH 7.4; and 0.2 mM PMSF)</li>
	<li>Check the efficiency of cleavage on a SDS-PAGE gel (Figure 5A).</li>
	<li>Load the cleaved MBP-Mgm101 by multiple injections on a Bio-Scale Mini Macro-Prep High S cartridge connected to a Bio-Rad Biologic DuoFlow FPLC system.</li>
	<li>Start the cation exchange chromatography by applying a step salt gradient of 5 mM, 300 mM, 500 mM, 750 mM and 1,000 mM of NaCl that is created by mixing Buffer A (5 mM NaCl; 10 mM Na-phosphate, pH 7.2; 1 mM PMSF) and Buffer B (1 M NaCl; 10 mM Na-phosphate, pH 7.2; 1 mM PMSF). Set the flow rate at 0.5 ml/min. Collect the Mgm101 fraction and check the purity of the protein on a SDS-PAGE gel (Figure 5B).</li>
</ol>
4. Size Exclusion Chromatography
<ol>
	<li>Combine the protein fractions from cation exchange chromatography. Dialyze the protein in GF equilibration buffer (20 mM MOPS, pH 7.0; 150 mM NaCl; 1mM EDTA, pH 7.4; 5 mM 2-Mercaptoethanol; 0.2 mM PMSF).</li>
	<li>Concentrate the protein with VIVASPIN 15R Ultrafiltration spin column to reduce the volume to ~1 ml by spinning at 3,000 x g at 4 &deg;C.</li>
	<li>Load Mgm101 on a calibrated Superose 6 prep grade column equilibrated with the chromatography buffer (20 mM MOPS, pH 7.0; 150 mM NaCl; 5 mM &beta;-mercaptoethanol; 1 mM EDTA, pH 7.4; 0.2 mM PMSF).</li>
	<li>Start the size exclusion with the chromatography buffer run at a flow rate of 0.5 ml/min.</li>
	<li>Collect the fractions of the purified Mgm101 peaks. Load aliquots of the fractions on a 12% SDS-PAGE for checking the final quality of the protein.</li>
	<li>Concentrate the protein with VIVASPIN 6, then quickly freeze the samples in liquid N2 and store at -80 &deg;C.</li>
	<li>Use the Mgm101 samples within 6-12 months for ssDNA-binding assay (Figure 8) and for structural visualization by transmission electron microscopy (Figure 9).</li>
</ol>

<ol>
	<li>San Filippo, J., Sung, P., Klein, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+eukaryotic+homologous+recombination.">Mechanism of eukaryotic homologous recombination.</a> Annu. Rev. Biochem. 77, 229-257 (2008).</li><li>Bishop, D. K., Park, D., Xu, L., Kleckner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DMC1:+a+meiosis-specific+yeast+homolog+of+E.+coli+recA+required+for+recombination,+synaptonemal+complex+formation,+and+cell+cycle+progression.">DMC1: a meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression.</a> Cell. 69, 439-456 (1992).</li><li>Shinohara, A., Ogawa, H., Ogawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rad51+protein+involved+in+repair+and+recombination+in+S.+cerevisiae+is+a+RecA-like+protein.">Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein.</a> Cell. 69, 457-470 (1992).</li><li>Passy, S. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Dmc1+protein+binds+DNA+as+an+octameric+ring.">Human Dmc1 protein binds DNA as an octameric ring.</a> Proc. Natl. Acad. Sci. U.S.A. 96, 10684-10688 (1999).</li><li>Story, R. M., Weber, I. T., Steitz, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+E.+coli+recA+protein+monomer+and+polymer.">The structure of the E. coli recA protein monomer and polymer.</a> Nature. 355, 318-325 (1992).</li><li>Yu, X., Jacobs, S. A., West, S. C., Ogawa, T., Egelman, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Domain+structure+and+dynamics+in+the+helical+filaments+formed+by+RecA+and+Rad51+on+DNA.">Domain structure and dynamics in the helical filaments formed by RecA and Rad51 on DNA.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 8419-8424 (2001).</li><li>Conway, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+a+Rad51+filament.">Crystal structure of a Rad51 filament.</a> Nat. Struct. Mol. Biol. 11, 791-796 (2004).</li><li>Bai, Y., Davis, A. P., Symington, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+allele+of+RAD52+that+causes+severe+DNA+repair+and+recombination+deficiencies+only+in+the+absence+of+RAD51+or+RAD59.">A novel allele of RAD52 that causes severe DNA repair and recombination deficiencies only in the absence of RAD51 or RAD59.</a> Genetics. 153, 1117-1130 (1999).</li><li>Bai, Y., Symington, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Rad52+homolog+is+required+for+RAD51-independent+mitotic+recombination+in+Saccharomyces+cerevisiae.">A Rad52 homolog is required for RAD51-independent mitotic recombination in Saccharomyces cerevisiae.</a> Genes Dev. 10, 2025-2037 (1996).</li><li>Paques, F., Haber, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+pathways+of+recombination+induced+by+double-strand+breaks+in+Saccharomyces+cerevisiae.">Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae.</a> Microbiol. Mol. Biol. Rev. 63, 349-404 (1999).</li><li>Lopes, A., Amarir-Bouhram, J., Faure, G., Petit, M. A., Guerois, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+novel+recombinases+in+bacteriophage+genomes+unveils+Rad52,+Rad51+and+Gp2.5+remote+homologs.">Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5 remote homologs.</a> Nucleic Acids Res. 38, 3952-3962 (2010).</li><li>Poteete, A. R., Sauer, R. T., Hendrix, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Domain+structure+and+quaternary+organization+of+the+bacteriophage+P22+Erf+protein.">Domain structure and quaternary organization of the bacteriophage P22 Erf protein.</a> J. Mol. Biol. 171, 401-418 (1983).</li><li>Passy, S. I., Yu, X., Li, Z., Radding, C. M., Egelman, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rings+and+filaments+of+beta+protein+from+bacteriophage+lambda+suggest+a+superfamily+of+recombination+proteins.">Rings and filaments of beta protein from bacteriophage lambda suggest a superfamily of recombination proteins.</a> Proc. Natl. Acad. Sci. U.S.A. 96, 4279-4284 (1999).</li><li>Ploquin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+structural+basis+for+a+bacteriophage+homolog+of+human+RAD52.">Functional and structural basis for a bacteriophage homolog of human RAD52.</a> Curr. Biol. 18, 1142-1146 (2008).</li><li>Chen, X. J., Guan, M. X., Clark-Walker, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MGM101,+a+nuclear+gene+involved+in+maintenance+of+the+mitochondrial+genome+in+Saccharomyces+cerevisiae.">MGM101, a nuclear gene involved in maintenance of the mitochondrial genome in Saccharomyces cerevisiae.</a> Nucl. Acids Res. 21, 3473-3477 (1993).</li><li>Meeusen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mgm101p+is+a+novel+component+of+the+mitochondrial+nucleoid+that+binds+DNA+and+is+required+for+the+repair+of+oxidatively+damaged+mitochondrial+DNA.">Mgm101p is a novel component of the mitochondrial nucleoid that binds DNA and is required for the repair of oxidatively damaged mitochondrial DNA.</a> J. Cell Biol. 145, 291-304 (1999).</li><li>Mbantenkhu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mgm101+is+a+Rad52-related+protein+required+for+mitochondrial+DNA+recombination.">Mgm101 is a Rad52-related protein required for mitochondrial DNA recombination.</a> J. Biol. Chem. 286, 42360-42370 (2011).</li><li>Nardozzi, J. D., Wang, X., Mbantenkhu, M., Wilkens, S., Chen, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+properly+configured+ring+structure+is+critical+for+the+function+of+the+mitochondrial+DNA+recombination+protein.">A properly configured ring structure is critical for the function of the mitochondrial DNA recombination protein.</a> Mgm101. J. Biol. Chem. 287, 37259-37268 (2012).</li><li>Zuo, X., Xue, D., Li, N., Clark-Walker, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+core+of+the+mitochondrial+genome+maintenance+protein+Mgm101p+in+Saccharomyces+cerevisiae+determined+with+a+temperature-conditional+allele.">A functional core of the mitochondrial genome maintenance protein Mgm101p in Saccharomyces cerevisiae determined with a temperature-conditional allele.</a> FEMS Yeast Res. 7, 131-140 (2007).</li><li>Itoh, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+packaging+proteins+Glom+and+Glom2+coordinately+organize+the+mitochondrial+nucleoid+of+Physarum+polycephalum.">DNA packaging proteins Glom and Glom2 coordinately organize the mitochondrial nucleoid of Physarum polycephalum.</a> Mitochondrion. 11, 575-586 (2011).</li><li>Janicka, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+RAD52-like+single-stranded+DNA+binding+protein+affects+mitochondrial+DNA+repair+by+recombination.">A RAD52-like single-stranded DNA binding protein affects mitochondrial DNA repair by recombination.</a> Plant J. 72, 423-435 (2012).</li></ol>

The authors declare that they have no competing financial interests.

It has been a challenge to produce a stable, native recombinant Mgm101 protein from E. coli possibly due to its insolubility in bacterial cells. In this report, we show that the MBP-fusion strategy allows the protein to be expressed at a reasonably high level. By using negative staining transmission electron microscopy and size exclusion chromatography, we have previously shown that the MBP-fusion protein forms uniform oligomers in vitro 18. It is possible that the folding and oligomerization of Mgm101 may be slower in the bacterial cells compared with the mitochondrial matrix. The unoligomerized Mgm101 may quickly form aggregates that are toxic in vivo. MBP-tagging may keep Mgm101 in a soluble conformation that allows productive oligomerization and diminishes its toxicity.
An important criterion for good quality Mgm101 preparation is to have minimal contamination by DNA. The A280/A260 ratio is used to estimate the level of contaminant DNA in the samples. Typically, a ratio &gt; 1.4 can be easily achieved, which is considered to be of good quality with &lt;0.75% of contaminating nucleic acids. If the A280/A260 ratio is below this level, extended digestion with DNAse I is recommended. In the future, it may be useful to see whether digestion with S1 nuclease and RNase can further reduce contaminating nucleic acids.
We have previously shown that storage of Mgm101 at 4 &deg;C for a few weeks induces the formation of long helical filaments 17. The mechanism of filament formation is not well understood. Over-filamentation affects the solubility of the protein. Therefore, it is strongly advised that aliquots of freshly prepared Mgm101 protein are quickly frozen in liquid nitrogen followed by storage at -80 &deg;C. The DNA binding activity of the protein remains unchanged after 6-12 months of storage under these conditions.
This protocol can also be used to prepare mutant proteins of Mgm101. The oligomerization state and the stability of Mgm101 are frequently affected by the mutations. Mild mutations may cause a partial precipitation of the purified proteins upon the cleavage from MBP. In these cases, increasing the culture volume to 6 liters may allow sufficient yield of the stable purified proteins from size exclusion chromatography. Severe mutations affecting oligomerization may allow the production of the proteins in a MBP-fused form, but the protein may not be stable after removal of MBP.
In summary, the current protocol is reliable which allows high level expression of large Mgm101 oligomeric rings in E. coli. This protocol may be adapted to the production of Mgm101 orthologs 20, 21 and other oligomeric proteins, especially when the solubility of those proteins is low in the bacterial cells.

Homologous recombination is important for the repair of double-strand breaks (DSBs) and interstrand crosslinks, and for the reinitiation of DNA replication from collapsed replication forks 1. In conventional homologous recombination, the central reaction is catalyzed by the ATP-dependent recombinases including RecA in prokaryotes, and Rad51 and Dmc1 in eukaryotes 1-3. These recombinases form nucleoprotein filaments on ssDNA, which are essential for initiating homology search and strand invasion within duplex DNA templates (Figure 1, left panel) 4-7. In addition to the conventional scheme, homologous recombination can also take place in a RecA/Rad51-independent manner (Figure 1, right panel). For instance, the yeast Rad52 and Rad59 proteins can directly catalyze the annealing of complementary ssDNA strands which are exposed by resectioning of dsDNA breaks. This recombination process, known as single strand annealing, generally does not involve homologous pairing with dsDNA templates. After annealing, heterologous tails are removed by exonucleases and nicks are ligated to restore genome continuity 8-10. Repair by the single strand annealing mechanism is often accompanied by deletions of genomic sequences between directly repeated regions.
Rad52 belongs to a diverse group of recombination proteins that are widespread among bacteriophages 11. These proteins are also known as Single Strand Annealing Proteins (SSAPs), based on their activity in promoting the annealing of homologous single stranded DNA molecules. The best characterized bacteriophage SSAPs are Red&beta; and Erf from the bacteriophages &lambda; and P22, RecT from the prophage rac and the Sak protein from the lactococcal phage ul36. The SSAPs are structurally characterized by a typical &beta;-&beta;-&beta;-&alpha; fold, although similarity is virtually undetectable in their primary sequences. They all form large homo-oligomeric rings of 10 - 14 fold symmetry in vitro 12-14. The functional implications of this characteristic higher order structural organization is not well understood.
We are interested in understanding the mechanism of homologous recombination in the mitochondrial genome. We have previously identified the MGM101 gene that is essential for the maintenance of mtDNA in Saccharomyces cerevisiae 15. MGM101 was subsequently found to be associated with mitochondrial nucleoids and is required for the tolerance of mtDNA to DNA-damaging agents 16. However, the study of Mgm101 has been held back in the last decade by the difficulty to produce recombinant Mgm101. We have recently succeeded in producing soluble Mgm101 at large quantities from E. coli using the MBP-fusion strategy. This has enabled us to demonstrate that Mgm101 shares biochemical and structural similarities with the Rad52-family of proteins 17,18. In this report, a three-step purification procedure is described, which produces homogeneous Mgm101for biochemical and structural analyses (Figure 2).

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Expression vector pMAL-c2E</td>
			<td>New England Biolabs</td>
			<td>#N8066S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PreScission Protease</td>
			<td>GE Healthcare Life Sciences</td>
			<td>#27-0843-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BL21-CodonPlus(DE3)-RIL cells</td>
			<td>Strategene</td>
			<td>#230245</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Roche Applied Science</td>
			<td>#11034626001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pepstatin</td>
			<td>Roche Applied Science</td>
			<td>#11359053001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phenylmethylsulfonyl fluoride (PMSF)</td>
			<td>Roche Applied Science</td>
			<td>#10837091001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Sigma</td>
			<td>#DN25-1G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Isopropyl &beta;-D-1-thiogalactopyranoside (IPTG)</td>
			<td>Roche Applied Science</td>
			<td>#11411446001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amylose resin</td>
			<td>New England Biolabs</td>
			<td>#E8021L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Econo-Column chromatography column</td>
			<td>BIO-RAD</td>
			<td>#7372512</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bio-Scale Mini Macro-Prep High S cartridge (1 ml)</td>
			<td>BIO-RAD</td>
			<td>#732-4130</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>VIVASPIN 15R Ultrafiltration spin column (10,000 MWCO)</td>
			<td>Sartorius Stedium</td>
			<td>#VS15RH02</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Superose 6 prep grade column</td>
			<td>Amersham Bioscirnces</td>
			<td>#17-0489-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>VIVASPIN 6 Ultrafiltration spin column (5,000 MWCO)</td>
			<td>Sartorius Stedium</td>
			<td>#VS0611</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

preparation of the mgm101 recombination protein by mbp-based tagging strategy

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the Mgm101 protein is involved in the recombinational repair of mitochondrial DNA. Recent investigations have indicated that Mgm101 is related to the Rad52-type recombination protein family. These proteins form large oligomeric rings and promote the annealing of homologous single stranded DNA molecules. However, the characterization of Mgm101 has been hindered by the difficulty in producing the recombinant protein. Here, a reliable procedure for the preparation of recombinant Mgm101 is described. Maltose Binding Protein (MBP)-tagged Mgm101 is first expressed in Escherichia coli. The fusion protein is initially purified by amylose affinity chromatography. After being released by proteolytic cleavage, Mgm101 is separated from MBP by cationic exchange chromatography. Monodispersed Mgm101 is then obtained by size exclusion chromatography. A yield of ~0.87 mg of Mgm101 per liter of bacterial culture can be routinely obtained. The recombinant Mgm101 has minimal contamination of DNA. The prepared samples are successfully used for biochemical, structural and single particle image analyses of Mgm101. This protocol may also be used for the preparation of other large oligomeric DNA-binding proteins that may be misfolded and toxic to bacterial cells.

Mgm101 is a Rad52-related recombination protein in mitochondria. Rad52 has been extensively studied for its role in mitochondrial DNA recombination (Figure 1). Recombinant Mgm101 can be prepared by a three-step procedure (Figure 2). This is facilitated by the use of the MBP-tagging strategy that allows Mgm101 to be expressed in a soluble form and subsequently released from the tag by proteolytic cleavage (Figure 3).
In a typical preparation, approximately 2-3 mg of MBP-Mgm101 fusion can be recovered from 1 liter of bacterial culture after amylose affinity chromatography. On a SDS-PAGE gel, MBP-Mgm101 is detected as a major band of ~70 kDa (Figure 4). Contamination by other proteins is minimal if the resin is appropriately washed before elution. After cleavage by PreScission Protease, MBP and Mgm101 are separated and shown as bands of 42 kDa and 28 kDa respectively on SDS-PAGE (Figure 5A). In the case of incomplete digestion, as indicated by the presence of a ~70 kDa band, an extended digestion with additional PreScission Protease is advised.
For the cation exchange chromatography of cleaved MBP-Mgm101, MBP is not retained by the column before the application of the salt (Figure 6). Typically, Mgm101 is eluted with 500 mM of salt without noticeable contamination by MBP (Figure 5B). A small peak at 300 mM is sometime visible, which corresponds to a trace amount of uncleaved MBP-Mgm101 fusion. It is possible that Mgm101 in this fraction is bound by contaminating DNA, which reduces binding affinity to the column. Thus, cation exchange is a necessary step to minimize DNA contamination.
Figure 7 shows the final step of Mgm101 purification by size exclusion chromatography. The Mgm101 oligomers typically elute in a monodispersed peak of ~400 kDa. Some mutant forms of Mgm101 are often seen with increased sizes between V0 (void volume) and the 400 kDa peak. The reason for this is still unknown. Mutations affecting the oligomerization of Mgm101 may induce the formation of aggregates which elute in the V0 fractions. However, we have never seen peaks corresponding to monomeric Mgm101. Mgm101 monomers are likely unstable in solution.
The purified Mgm101 has a high binding affinity to ssDNA (Figure 8). The binding affinity for ssDNA can be estimated based on the titration of the free ssDNA on the gel. The Mgm101/ssDNA complex migrates poorly out of the wells. A possible explanation is that Mgm101 is positively charged and the binding of multiple subunits of Mgm101 to ssDNA neutralizes its negative charges which makes the protein/DNA complexes immobile under the electrophoresis conditions. When appropriately prepared, Mgm101 binds ssDNA with a Kd of ~200 nM.
Figure 9 shows the direct structural visualization of the purified Mgm101 by negative stain transmission electron microscopy. Freshly prepared Mgm101 is mainly present as large oligomeric rings with a diameter of ~20 nm. Instead, the aged Mgm101 samples form compressed filaments. These filaments are not formed by the simple stacking of the rings. There is clearly a helical pattern in the filaments. The conditions that modulate the filamentation process are currently under investigation. It is advisable that the protein quality is checked on SDS-PAGE after the incubation at 4 &deg;C for the aged samples before being analyzed by electron microscopy.
<img alt="Figure 1" src="/files/ftp_upload/50448/50448fig1.jpg" /> 
Figure 1. Simplified schematic diagram showing that Rad52 serves as a mediator for the loading of the Rad51 recombinase on the recombination site in a canonical homologous recombination pathway (left panel), and promotes single strand annealing independent of Rad51 (right panel).
<img alt="Figure 2" src="/files/ftp_upload/50448/50448fig3.jpg" fo:content-width="4in" fo:src="/files/ftp_upload/50448/50448fig3highres.jpg"/> 
Figure 2. Overall scheme for the preparation of recombinant Mgm101. Mgm101 is first expressed in E. coli in a MBP-fused form. Bacterial cells are lysed by sonication. DNAse I digestion is applied to reduce DNA contamination. The MBP-Mgm101 fusion is then purified from the DNA-depleted lysate by using an amylose column. After proteolytic cleavage, Mgm101 is released and separated from MBP by cation exchange chromatography. This step is necessary to further reduce contamination by DNA. The eluted Mgm101 fraction is then purified to homogeneity by passing it through a Superose 6 prep grade column.
<img alt="Figure 3" src="/files/ftp_upload/50448/50448fig2.jpg" fo:content-width="5in" fo:src="/files/ftp_upload/50448/50448fig2highres.jpg"/> 
Figure 3. Physical map of the Mgm101-expressing plasmid pMAL-c2E-MGM101. The cleavage site for the PreScission protease between MBP and Mgm101 is indicated. The proteolytic cleavage yields MBP and Mgm101 with a molecular weight of 42 and 28 kDa respectively.
<img alt="Figure 4" src="/files/ftp_upload/50448/50448fig4.jpg" /> 
Figure 4. SDS-PAGE showing the purity of the MBP-Mgm101 fusion protein after amylose affinity chromatography. Protein concentration is determined by measuring the absorbance at 280 nm and approximately 5 &mu;g of Mgm101 is loaded on the gel. The gel is stained with coomassie blue.
<img alt="Figure 5" src="/files/ftp_upload/50448/50448fig5.jpg" /> 
Figure 5. SDS-PAGE analysis of Mgm101. A) The release of Mgm101 from the MBP-Mgm101 fusion protein after cleavage with the PreScission protease. After digestion with the protease O/N, an aliquot containing approximately 5 &mu;g of protein is loaded on the gel. The gel is stained by coomassie blue. Mgm101 migrates slightly slower than its expected size of 28 kDa, due to the overall positive charge of the protein under the standard SDS-PAGE condition. B) Mgm101 after separation from MBP by cation exchange chromatography.
<img alt="Figure 6" src="/files/ftp_upload/50448/50448fig6.jpg" /> 
Figure 6. Chromatogram showing the separation of Mgm101 from MBP by cation exchange chromatography. The cleaved MBP-Mgm101 is injected into a Bio-Scale Mini Macro-Prep High S cartridge multiple times. A step salt gradient of 5 mM, 300 mM, 500 mM, 750 mM and 1,000 mM of NaCl is then applied. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50448/50448fig6large.jpg" target="_blank"> Click here to view larer figure</a>.
<img alt="Figure 7" src="/files/ftp_upload/50448/50448fig7.jpg" /> 
Figure 7. Chromatogram showing the elution profile of Mgm101 oligomers on a calibrated Superose 6 prep grade column. Chromatography is performed by at a flow rate of 0.5 ml/min. The eluted Mgm101 is observed as a peak of ~400 kDa. V0: void volume. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50448/50448fig7large.jpg" target="_blank"> Click here to view larer figure</a>.
<img alt="Figure 8" src="/files/ftp_upload/50448/50448fig8.jpg" /> 
Figure 8. Electrophoretic mobility shift assay showing that the purified Mgm101 has a robust binding activity to ssDNA. The 1.25 x 10-3 &mu;M of [&gamma;-32P]ATP-labeled oligonucleotide HS40AT-F (5'-TTAATATTTAATAATATAATCAATAAATAATATTATAATAATATAATATAA-3') is mixed with Mgm101 at the indicated concentrations in the binding buffer of 20 mM Tris-HCl, pH 8.0; 100 mM KCl; 5 mM MgCl2; 1 mM EDTA, pH 8.0; 10% glycerol; 1 mM DTT; 50 &mu;g/ml BSA; and 1 mM PMSF. After incubation at RT for 30 min, the samples are loaded onto a 6% native acrylamide gel and run in 0.5X TBE buffer at 4 &deg;C.
<img alt="Figure 9" src="/files/ftp_upload/50448/50448fig9.jpg" /> 
Figure 9. Electron micrographs showing that the structural organization of fresh (left panel) and aged (right panel) Mgm101. Negative staining transmission electron microscopy is carried out using a JEM-2100 transmission electron microscope (JEOL) operating at 200 kV. For negative stain of aged Mgm101, the protein is stored at 4 &deg;C in the GF buffer (20 mM MOPS, pH 7.0; 150 mM NaCl; 5 mM &beta;-mercaptoethanol; 1 mM EDTA, pH 7.4; and 0.2 mM PMSF) for 4 weeks before being analyzed. (Courtesy of Dr. Stephan Wilkens).

Watch this Scientific Journal Video about Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy at JoVE.com

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

The yeast mitochondrial nucleoid protein, Mgm101, is a Rad52-type recombination protein that forms large oligomeric rings. A protocol is described to prepare soluble recombinant Mgm101 using the Maltose Binding Protein (MBP)-tagging strategy coupled with cation exchange and size exclusion chromatography.

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the ...

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12.0K Views.  State University of New York Upstate Medical University. The yeast mitochondrial nucleoid protein, Mgm101, is a Rad52-type recombination protein that forms large oligomeric rings. A protocol is described to prepare soluble recombinant Mgm101 using the Maltose Binding Protein (MBP)-tagging strategy coupled with cation exchange and size exclusion chromatography.

Video: Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

Predicting the Effectiveness of Population Replacement Strategy Using Mathematical Modeling

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy.  Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies.  The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy. Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies. The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy.  ...

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo5. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-1116,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands8. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.

A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.

Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments.

Random microseed matrix screening  (rMMS) is a protein crystallization technique in which seed crystals are added  to random screens. By increasing the ...

Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends

Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are thought to play a vital role in the microtubule-dependent chromosome motions during cell division, but the relevant proteins and their exact roles are ill-defined. Thus, there is a growing need to develop assays with which to study such motility in vitro using purified components and defined biochemical milieu. Microtubules, however, are inherently unstable polymers; their switching between growth and shortening is stochastic and difficult to control. The protocols we describe here take advantage of the segmented microtubules that are made with the photoablatable stabilizing caps. Depolymerization of such segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting studies of motility at the disassembling microtubule ends. This technique can be used to carry out a quantitative analysis of the number of molecules in the fluorescently-labeled protein complexes, which move processively with dynamic microtubule ends. To optimize a signal-to-noise ratio in this and other quantitative fluorescent assays, coverslips should be treated to reduce nonspecific absorption of soluble fluorescently-labeled proteins. Detailed protocols are provided to take into account the unevenness of fluorescent illumination, and determine the intensity of a single fluorophore using equidistant Gaussian fit. Finally, we describe the use of segmented microtubules to study microtubule-dependent motions of the protein-coated microbeads, providing insights into the ability of different motor and nonmotor proteins to couple microtubule depolymerization to processive cargo motion.

Microtubules are inherently unstable polymers, and their switching between growth and shortening is stochastic and difficult to control. Here we describe protocols using segmented microtubules with photoablatable stabilizing caps. Depolymerization of segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting analysis of motions with the disassembling microtubule ends.

Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein subcellular localization changes; in particular, a nuclear translocation under a heat stress condition. Proteins react correspondingly to external and internal signals. A common mechanism is to change its subcellular localization. This article describes a protocol to track protein localization that does not require an antibody, radioactive labeling, or a confocal microscope. In this article, GFP is used to tag the target protein EXL-1 in C. elegans, a member of the chloride intracellular channel proteins (CLICs) family, including mammalian CLIC4. An integrated translational exl-1::gfp transgenic line (with a promoter and a full gene sequence) was created by transformation and &#947;-radiation, and stably expresses the gene and gfp. Recent research showed that upon heat stress, not oxidative stress, EXL-1::GFP accumulates in the nucleus. Overlapping the GFP signal with both the nuclei structure and the DAPI signals confirms the EXL-1 subcellular localization changes under stress. This protocol presents two different fixation methods for DAPI staining: ethanol fixation and acetone fixation. The DAPI staining protocol presented in this article is fast and efficient and preserves both the GFP signal and the protein subcellular localization changes. This method only requires a fluorescence microscope with Nomarski, a FITC filter, and a DAPI filter. It is suitable for a small laboratory setting, undergraduate student research, high school student research, and biotechnology classrooms.

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

This protocol demonstrates how to track a protein nuclear translocation under heat stress by using a green fluorescence protein (GFP) fusion protein as a marker and 4',6-diamidino-2-phenylindole (DAPI) staining. The DAPI staining protocol is fast and preserves the GFP and protein subcellular localization signals.

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

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Predicting the Effectiveness of Population Replacement Strategy Using Mathematical Modeling

Detection of Protein Ubiquitination

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry