The overall goal of this procedure is to fractionate a Durant and non-ad durant mammalian cells in situ U in order to examine protein localization. This is accomplished by first attaching the cells to microscope cover slips. The second step of the procedure is to remove the cytoplasmic and loosely held nuclear proteins.
The third step of the procedure is to remove the tightly held nuclear proteins. The final step of the procedure is to remove the chromatin fraction, leaving the nuclear matrix and dissociated proteins on the cover slip. Ultimately, results can be obtained that show protein subcellular localization and colocalization through immunofluorescence microscopy.
Hi, I am Anya Ho Hai from the laboratory of Kevin Gaston in the School of Biochemistry at the University of Bristol. Today we'll show you a procedure for institute subcellular fractionation. We use this procedure to study the localizations of transcription factors.
So let's get started. We will begin by demonstrating the procedures for attaching both non-ad adherent and a adherent mammalian cells to cover slips. Here the non-adherence cells are K 5 6 2 cells and the A adherent cells across seven cells.
To attach non-adherence cells place a polyol lysine coated cover slip in a well of a six well plate with the coated surface facing up seed. K 5 62 cells are a density of seven times percent of the five cells per well. In KO's modified eagle's medium supplemented with 10%fetal bovine serum and penicillin streptomycin.
Unlike non-adherence cells, adherence cells generally attach well onto normal microscope. Cover slips after two washes in PBS detach the cost seven cells by digesting with trypsin in PBS at 37 degrees Celsius for two to five minutes. It is important not to over digest the cells and to stop the digestion as soon as predominantly individual floating cells are present.
Stop the digestion by seeding the cells at a density of three times 10 of the five cells per well. In DMEM supplemented with 10%FBS and PSG on a normal uncoated cover slip. In a six well plate incubate both the non-ad adherent and a adherent cells for 24 hours at 37 degrees Celsius and 5%carbon dioxide.
On the day of the subcellular fractionation, prepare and filter fresh cytoskeleton buffer. Remove from the incubator the cells seeded onto cover slips yesterday. In this demonstration, we will fractionate cost seven cells.
Pour off the media and wash the cells twice with ice cold phosphate buffered saline. Use the cells from one cover slip to prepare a whole cell extract for western blotting gently place 200 microliters of TES buffer directly onto the cover. Slip and incubate for one or two minutes.
At 22 degrees Celsius, the whole cell extract is then removed and the proteins precipitated by adding 250 microliters of one molar ammonium sulfate and incubating on ice. Since additional western samples will be prepared in parallel with each fraction, leave the sample on ice until all the other samples are ready. Wash the remaining cover slips with one milliliter of ice cold PBS twice at 22 degrees Celsius.
To avoid losing cells during the washing steps, add the PBS slowly to the side of each well. Avoiding the cover slip. Tilt the plate carefully two or three times.
Remove the cover slip representing whole cells and transfer to a fresh six. Well plate. Fix the cells by gently adding one milliliter of 4%for formaldehyde in PBS incubate at four degrees Celsius for 30 minutes.
Gently remove the PBS from the remaining wells by slow pipetting. Extract the cytoplasmic and loosely held nuclear proteins by gently placing 200 microliters of CSK buffer plus 0.1%Triton X 100 directly onto each remaining cover slip. After incubating on ice for one minute, remove the extract and prepare a western sample as shown earlier.
Next, wash the cover slips carefully with one milliliter of ice core PBS twice as shown earlier. Remove the cover slip representing tightly held nuclear material and transfer to a fresh six Well plate. Fix the cells by adding 4%formaldehyde in PBS and incubating at four degrees Celsius.
Gently remove the PBS from the remaining wells. Extract the tightly held proteins by gently placing 200 microliters of CSK buffer plus 0.5%Triton X 100 directly onto each remaining cover. Slip and incubating on ice for 20 minutes.
After preparing a Western sample from the extract, wash the cover slips with one milliliter of ice cold PBS twice. Now remove the cover slip representing the chromatin fraction and fix the cells. As before.
Gently remove the PBS from the remaining wells. Then place 200 microliters of CSK buffer plus 100 micrograms per milliliter of DNAs one onto the remaining cover slips. This time incubate for 30 minutes at 37 degrees Celsius.
To prepare chromatin extract for western blotting, remove the chromatin extract for western blotting and precipitate as shown previously. After washing the remaining cover slips with ice cold PBS twice, remove the cover slip representing the nuclear matrix fraction and fix the cells. A nuclear fraction for western blotting can be obtained by adding TES buffer to an additional cover slip.
As previously described, when all cell fractions have been collected and fixed, they can be processed for immunofluorescence microscopy. Start this procedure by washing each cover slip gently two to three times with one milliliter of PBS at 22 degrees Celsius. Next, add 400 microliters of 0.1%Triton X 100 in PBS to each cover, slip and incubate at 22 degrees Celsius for 10 minutes.
After 10 minutes, wash each cover slip gently two to three times with one milliliter of PBS at 22 degrees Celsius. Then add 400 microliters of 3%bovine serum albumin in PBS and incubate at 22 degrees Celsius for 20 minutes. This will reduce potential background staining.
Wash each cover slip gently two to three times with PBS and then place 100 microliters of the primary antibody directly onto the cover. Slip incubate at 22 degrees Celsius for one hour. After one hour.
Wash each cover slip with PBS and place 200 microliters of secondary antibody onto the cover. Slip incubate 22 degrees Celsius for one hour away from light to mount each cover slip onto a glass microscope. Slide is vector shield mounting medium containing dappy.
Wipe excess mounting medium from around the cover slip using a paper towel. Incubate the slide at 22 degrees Celsius for at least 30 minutes away from light. When the incubation is complete, fix the cover slip to the glass slide using clear acrylic nail polish.
Keep the slides away from light before visualization by fluorescence microscopy shown here a representative results from a successful fractionation of unresected cost seven cells fractionated cost. Seven cells were fixed with formaldehyde and immunostain using anti tubulin, anti hiss stone H one or anti Lamin HC antibodies conjugated to trixy followed by treatment with mounting medium containing dpi. This technique is well suited to the detection of GFP fusion proteins as shown in this subcellular fractionation of COS seven cells expressing GFP fued for the human papillomavirus type six E two protein.
The merged images reveal that some of the GFP six E two protein is associated with the nuclear matrix. We have just shown you how to fractionate the MA cells in situ two. When doing this procedure, it's important to remember to handle the washing step with care.
So that's it. Thanks for watching and good luck with your experiment.
