This work was supported by NIH grants ARRA RO3 AI078145 and 1SC3GM113743 to RZ.

1. Cell Extract Preparation
<ol>
	<li>Grow the Leishmania cells in a sterile plastic bottle sealed with air tight cap at 26 &#186;C in a medium made of 1x M199 supplemented with 20 mM HEPES pH7.4, 100 U/ml penicillin, 100 &#181;g/ml streptomycin, 5 &#181;g/ml heme, 0.35 g/L NaCO2H, 0.1 mM adenine, and 2 &#181;g/ml biopterin without shaking. Harvest the cells by centrifugation at 1,500 g for 5 min at 4 &#186;C when they reach a cell density of 1-2 x 107/ml.</li>
	<li>Discard the supernatant with a pipette and wash the cells by resuspending the cell pellet with a serological pipet in half of the culture&#39;s volume of cold phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH7.4). Dispose cell supernatants according to BL2 safety guidelines.</li>
	<li>Centrifuge cells again at 1,500 g for 5 min at 4 &#186;C. Discard the supernatant with a pipette. Proceed to the next step or snap freeze the cell pellet in liquid nitrogen for long term storage at -80 &#186;C (up to three months).</li>
	<li>Prepare 2x lysis buffer (0.5 M sucrose, 0.1 M TrisHCl, pH7.5, 2 mM EDTA, and 2x protease inhibitor cocktail) and keep it at 4 &#186;C on ice.</li>
	<li>Resuspend the cell pellet (fresh or frozen) in equal volume of 2x lysis buffer. Add 1x volume of glass beads. Vortex vigorously at 4 &#186;C for 10 min.</li>
	<li>Add 2 volumes of 1x lysis buffer and mix. Centrifuge cell extracts at 1,500 x g at 4 &#186;C for 10 min to pellet unbroken cells and nuclei.</li>
	<li>Transfer the supernatant with a pipet into a fresh cool centrifuge tube and keep cell extracts on ice until completion of the experiment.</li>
</ol>
2. Determine the Protein Concentration of the Cell Extract Using Protein Estimation Kit Such as Bicinchoninic Acid Assay
<ol>
	<li>Prepare the bicinchoninic acid (BCA) solution (1 ml/tube) by mixing the BCA and copper (II) sulfate in a ratio of 49:1 (v/v).</li>
	<li>Prepare the protein standards of 0, 10, 20, 30, 40, 50, and 60 &#181;g/ml by diluting a 10 mg/ml bovine serum albumin (BSA) stock solution into 1 ml aliquots of the BCA solution.</li>
	<li>Add 2 &#181;l of cells extracts in 1 ml of BCA solution in duplicates. Incubate standards and protein samples for 10 min in a pre-warmed 60 &#176;C water bath.</li>
	<li>Transfer samples to ice for 3 min. Measure the absorbance of the standards and the protein samples with a spectrophotometer at a wavelength of 562 nm.</li>
	<li>Calculate the protein concentration of the cell extracts using the BSA standard as a reference as described in the manufacturer&#39;s protocol. Dilute cell extracts to a protein concentration of 10 mg/ml with 1x lysis buffer.</li>
</ol>
3. Enzymatic Assay in 200 &#956;l per Tube
NOTE: Carry the following steps in a chemical hood.
<ol>
	<li>Test each sample in duplicate in a 15 ml conical tube. Prepare 20 &#956;l 1 M TrisHCl pH 7.5 per tube and keep it on ice. Prepare 2 ml of chloroform/methanol (1:1 (v/v); stopping solution) at RT for each tube.</li>
	<li>Pipet 20 &#956;l of 1 M TrisHCl pH 7.5 in each 15 ml conical tube on ice.</li>
	<li>Follow radiation safety guidelines from here on. Add the equivalent of 0.06 &#956;M (0.2 &#956;Ci) S-[Methyl-3H]adenosyl-L-methionine and 50 &#956;M cold SAM per tube for a total of 50.06 &#956;M of SAM. Add x &#956;l of cold water where x = 200-(20 (for buffer) + 20 (for cell extracts) + volume of cold and radioactive SAM) per tube.</li>
	<li>Transfer each conical tube to a pre-warmed 30 &#186;C water bath. Add to each tube 20 &#956;l of cell extracts (equivalent of 200 &#956;g of protein) to start the reaction. Incubate for the desired time (0 to 45 min).</li>
	<li>Stop the reaction by adding 2 ml of chloroform/methanol (1:1; v/v; stopping solution) to each tube. Transfer the conical tube to RT (20-25 &#186;C).</li>
</ol>
4. Lipid Extraction
NOTE: Carry the following steps in a chemical hood.
<ol>
	<li>Add 700 &#956;l of water to each tube containing the enzymatic reaction sample. Vortex vigorously for 30 sec. Centrifuge at 1,500 x g for 5 min at RT to separate the organic from the water phase.</li>
	<li>Transfer the lower organic phase into a new 15 ml conical tube with a pipette. Add 1 ml of water to each &#34;lower phase&#34; containing tube and vortex vigorously for 30 sec. Centrifuge again at 1,500 x g for 5 min to separate the organic from the water phase.</li>
	<li>Transfer the lower organic phase into a scintillation tube with a pipette. Dry samples under a stream of N2. Dispose the water phases containing the non-incorporated radioactive SAM and the radioactive conical tubes as per radiation guidelines.</li>
	<li>Add 2 ml/tube of scintillation liquid. Measure the incorporated radioactivity with a scintillation counter according to the manufacturers&#39; protocol and instrument&#39;s use.</li>
	<li>Calculate the enzymatic activity in nmol/mg protein using the following general equation: 
	cpm value x 103 x [total (radioactive and cold) concentration of SAM (mM)] x 5 specific activity of radioactive SAM (Ci/mmol) x [concentration of radioactive SAM (mM)] 
	NOTE: the above protocol can be altered for a SAM concentration dependent or a protein dependent PEMT assay. For SAM concentration dependent enzymatic assay, time is kept constant (15 min, which is in the linear range) and various amounts of cold SAM are added to the assay while for protein dependent PEMT assay, SAM concentration (we chose 0.05 mM) and time are kept constant (15 min). Also the pH of the buffer can be changed as needed if some PEMT enzymes have a different optimal pH value.</li>
</ol>

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No conflict of interest declared

This simple, quick PEMT assay allows the quantification of methylated forms of PE that results from the transfer of radioactive methyl groups from SAM onto PE using whole cell extract as a protein source. It is fast, sensitive, reproducible, and also suitable for purified enzymes 17. Monomethyl- or dimethyl-PE can be added to the assay if the methyltransferase of interest is specific to these substrates rather than to PE 12,13,18,19. If purified PEMT enzyme is used, PE can be added to the assay. A limitation of this protocol is that the assay does not identify the reactions products (monomethyl-PE, dimethyl-PE, or PC). However, the identity of the reaction products (monomethyl-PE, dimethyl-PE, PC) can further be analyzed by one dimensional thin layer chromatography as described in 20,21. Further, some of the SAM degradation products such as S-adenosylhomocysteine and 5&#39;-methylthioadenosine may inhibit PEMT activity by feedback inhibition. However, Leishmania possesses an S-adenosylhomocysteine hydrolase 22, which cleaves S-adenosylhomocysteine into adenine and S-ribosylhomocysteine, and a methylthioadenosine phosphorylase, which produces adenine and methylthioribose-1-phosphate 23. However, it is not known whether S-adenosylhomocysteine hydrolase and methylthioadenosine phosphorylase activities are high enough to efficiently metabolize S-adenosylhomocysteine and methylthioadenosine, respectively, so that no inhibition of PEMT activity occurs. In the instance S-adenosylhomocysteine hydrolase and/or 5&#39;-methylthioadenosine metabolic enzyme are absent in the cell of interest, addition of the respective purified, recombinant enzymes can be added to the assay to relieve feedback inhibition by SAM degradation products 24,25,26.
There are four critical steps in this protocol: i) the protease inhibitor cocktail powder has to be added to the lysis buffer just before use (step 1.1); ii) the whole cell extracts are to be utilized within the following hours after preparation (after step 1.4); iii) during the lipid extraction steps (steps 4.2 and 4.4), caution needs to be applied to not transfer any of the interphase or water phase, which contains the excess tritiated SAM, and iv) to aliquot the radioactive S-[Methyl-3H]adenosyl-L-methionine and cold reagent upon receipt as repeated cycles of freeze and thaw degrade it into 5&#39;-methylthioadenosine and homoserine lactone followed by hydrolysis to adenine and S-pentosylmethionine 26,27,28, which may account for lack of measurable PEMT activity. Bad whole cell extracts may also be responsible for no enzymatic activity. In this case, the quality of the cell extract can be assessed by measuring another known enzymatic activity. Last, lack of PEMT activity may rely on the PEMT enzyme being specific to monomethyl-PE or dimethyl-PE rather than PE. Addition of monomethyl-PE and/or dimethyl-PE substrates to the assay may restore PEMT activity.
An alternative, non-radioactive SAM dependent methyltransferase assay has been developed, that relies on the enzymatic quantification of homocysteine, the transmethylation products of SAM 29. This protocol has been successfully applied to measure PE specific methyltransferase activity 16. Unfortunately, this assay is only suitable for purified enzyme and not for whole cell extract, and does not reveal the identity of the reaction products either.
The present protocol can possibly be applied to any cell type. Additionally, this assay can be used to test potential drugs specific to a PEMT of interest in the context of testing new anti-microbial compounds to fight bacterial infections or novel therapeutics against diet-induced atherosclerosis, obesity, and insulin resistance.

Phosphatidylethanolamine methyltransferase (PEMT) enzymes catalyze the covalent attachment of one or more methyl groups using S-adenosylmethionine (SAM) as the methyl group donor onto PE, monomethyl-PE or dimethyl-PE to give monomethyl-PE, dimethyl-PE and/or phosphatidylcholine (PC). These enzymes are almost ubiquitous in animal cells and fungi. They can also be found in some plants 1 and approximately 10% of bacteria, particularly those that interact with eukaryotes 2.
PEMTs are relevant to the biology of the cell not only by contributing to the production of PC, which is the main lipid class in animal cells, but also by fulfilling other important cellular functions. In mammals, PEMTs are mainly expressed in the liver where they are required for normal secretion of very low-density lipoprotein and they also contribute to diet-induced obesity 3, atherosclerosis 4, and insulin resistance 5. Additionally, mammalian PEMT are also expressed in adipocytes, although to lower levels, and participate in fat deposition 6,7. PEMT role in cancer development 8, apoptosis 9, and cell growth 10 have also been demonstrated. In bacteria, PEMT enzymes have been shown to be important for normal cell growth 2, virulence 2, and symbiosis with the host plant 11.
The goal and rationale of the present protocol is to measure PEMT activity from whole cell extracts without the need to purify the enzyme. Two distinct protocols have been developed to measure PEMT activity. The first and most common one measures the transfer of tritiated methyl group from radioactive SAM onto PE, which is the topic of this article. This protocol has been originally developed to measure PEMT activity from yeast 12 and mammalian cells (liver) 13 to gain an understanding of PC biosynthesis in these cells as well as to determine the specificity of these enzymes. Later, this technique has been applied to other cell types such as bacteria 2 (using a basic pH value for the assay though 15) and protozoan parasites 14. This technique can be used with whole cell extracts as well as purified enzyme, and can potentially be applied to any cell extract system. A non radioactive assay has also been designed that relies on the enzymatic quantification of S-adenosylhomocysteine, the transmethylation product of SAM 16. The latter assay may be more convenient as it does not involve radioactivity but it is only suitable for purified enzymes.

<table border="0" cellpadding="0" cellspacing="0" width="630">
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">S-[Methyl-3H]adenosyl-L-methionine (specific activity of 5-15 Ci/mMole)</td>
			<td style="width:107px;">Perkin Elmer</td>
			<td style="width:119px;">NET155050UC</td>
			<td style="width:188px;">Aliquot the reagent and freeze at -20 &#176;C; follow radiation safety guidelines while using this reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Protease inhibitor cocktail</td>
			<td style="width:107px;">Roche Life Sciences</td>
			<td style="width:119px;">11836170001</td>
			<td>dilute it fresh&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass beads, acid washed, 425-600 mm</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td>G8772</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bicinchoninic acid solution</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td style="width:119px;">B9643</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Copper (II) sulfate</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td style="width:119px;">C2284</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Scintillation counter MicroBeta2 with 1-detector</td>
			<td style="width:107px;">Perkin Elmer&#160;</td>
			<td style="width:119px;">2450-0010</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spectrophotometer Biomate 3</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td>840208300</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BSA stock solution (10 mg/ml)</td>
			<td style="width:107px;">New England Biolabs</td>
			<td style="width:119px;">B9001S</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Scintillation liquid&#160;</td>
			<td style="width:107px;">Research Product International Corp</td>
			<td style="width:119px;">111198</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">S-(5&#39;-Adenosyl)-L-methionine chloride (hydrochloride)&#160;</td>
			<td style="width:107px;">Cayman Chemicals</td>
			<td>13956</td>
			<td style="width:188px;">dilute the reagent in 20 mM HCl and freeze aliquots at -80 &#176;C&#160;</td>
		</tr>
	</tbody>
</table>

in vitro assay to measure phosphatidylethanolamine methyltransferase activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

Figure 1 shows a time dependant PEMT assay, which was carried out with Leishmania whole cell extract as an enzyme source using endogenous PE as a substrate. The amount of radioactivity in the organic phase was quantified by scintillation counting. The resulting numbers were utilized to calculate the amount of tritiated methyl groups transferred onto PE. The PEMT activity was linear for approximately 20 min. It then reached a plateau at around 30 min, after which it stayed constant for another 15 min. As expected, PEMT activity was not detected when no cell extracts were added to the assay (Figure 2). Further, this activity was abolished in the presence of 100 &#956;M octadecyltrimethylammonium bromide, which is an inhibitor of L. major PE methyltransferases LmjPEM1 and LmjPEM2 14. PEMT activity was also protein concentration dependent, and this activity was linearly proportional to the amount of protein applied for the enzymatic assay (Figure 3). Lastly, a SAM concentration dependent PEMT assay was carried out, in which increasing concentrations of SAM were tested (Figure 4). PEMT activity reached a plateau at SAM concentration of approximately 0.5 mM. All together, these four assays demonstrate that PEMT activity is specific and can be measured from whole cell extracts without the need to purify the enzyme(s).
<img alt="Figure 1" src="/files/ftp_upload/53302/53302fig1.jpg" />
Figure 1.&#160;Time dependent PEMT assay. The enzymatic assay was performed twice in duplicate with 0.2 mg of whole Leishmania cell extracts as a function of time. PEMT activity is represented as nmol methyl groups transferred onto PE per mg of protein and per hr. For time &#34;O&#34;, cell extracts were first mixed with 2 ml of stopping solution made of chloroform/methanol (1:1, by volume) before being added to the assay solution containing SAM. Standard deviations are shown.
<img alt="Figure 2" src="/files/ftp_upload/53302/53302fig2.jpg" />
Figure 2. Specificity of PEMT activity. PEMT assay was carried out twice in duplicate for 15 min in the presence of 0.05 mM SAM. 1, 0.2 mg protein extract; 2, no cell extract; 3, 0.2 mg protein and 0.1 mM octadecyltrimethylammonium bromide. Standard deviations are shown.
<img alt="Figure 3" src="/files/ftp_upload/53302/53302fig3.jpg" />
Figure 3. Protein dependent PEMT assay. The enzymatic assay was performed twice in duplicate in the absence (point &#39;O&#39;) or presence of various amounts of Leishmania proteins with 0.05 mM SAM for 15 min. Standard deviations are shown.
<img alt="Figure 4" src="/files/ftp_upload/53302/53302fig4.jpg" />
Figure 4. The PEMT assay was carried out twice in duplicate with 0.2 mg of whole Leishmania cell extracts in the presence of various concentrations of SAM for 15 min. Standard deviations are shown.

Watch this Scientific Journal Video about In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity at JoVE.com

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from ...

in-vitro-assay-to-measure-phosphatidylethanolamine-methyltransferase

Nanyang Technological University

Research

JoVE Journal

Biology

9.8K Views.  St John's University. The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine. 

Video: In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively inert because it has two unpaired electrons located in different pi * anti-bonding orbitals. These two electrons have parallel spins, meaning they rotate in the same direction about their own axes. This is why the oxygen molecule is not very reactive. Activation of oxygen may occur by two different mechanisms; either through reduction via one electron at a time (monovalent reduction), or through the absorption of sufficient energy to reverse the spin of one of the unpaired electrons. This results in the production of reactive oxidative species (ROS). There are a number of ways in which the human body eliminates ROS in its physiological state. If ROS production exceeds the repair capacity, oxidative stress results and damages different molecules. There are many different methods by which oxidative stress can be measured. This manuscript focuses on one of the methods named cell gel electrophoresis, also known as &#8220;comet assay&#8221; which allows measurement of DNA breaks. If all factors known to cause DNA damage, other than oxidative stress are kept constant, the amount of DNA damage measured by comet assay is a good parameter of oxidative stress. The principle is simple and relies upon the fact that DNA molecules are negatively charged. An intact DNA molecule has such a large size that it does not migrate during electrophoresis. DNA breaks, however, if present result in smaller fragments which move in the electrical field towards the anode. Smaller fragments migrate faster. As the fragments have different sizes the final result of the electrophoresis is not a distinct line but rather a continuum with the shape of a comet. The system allows a quantification of the resulting &#8220;comet&#8221; and thus of the DNA breaks in the cell.

Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

Measuring In Vitro ATPase Activity for Enzymatic Characterization

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary.

Measuring In Vitro ATPase Activity for Enzymatic Characterization

We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate ...