I want to perform Co-IP from the bovine kidney cell line transfected with the Myc-tagged bait and HA-tagged prey proteins. I am using Pierce RIPA lysis buffer for the cell lysis but the lysate I'm getting is very sticky and the protein content is very low wen seen with SDS-PAGE.
the complete protocol as follows:
1.Seeding of the 6-well plate with the bovine kidney cells24 h before transfection with both the plasmids.
2. Co-transfection of plasmids using lipofectamine-2000.
3.after 48 h, decanting of the culture medium.
4.washing of cells 2X with chilled PBS.
5. addition of 400 µl of chilled RIPA buffer per well.
6. Incubation on ice for 10 min.
7. Scraping of cells and centrifugation of lydate at 13000 rpm for 30 min at 4ºC
8. after centrifugation the lysate is very viscous and cell plate ususally come along with supernatant. Kindly suggest
Hello, the viscosity is related with DNA. You should put DNAasas in the final buffer to break this DNA. I hope your comments
I have added benzonase to the lysate but still the band profile on SDS-page is very weak. It seems to me as the cells are not completely lysed.
This viscosity is usually due to DNA. You could try passing the lysate through a fine needle and syringe e.g. 23G 10 times to try and lyse cells better. Secondly, you could sonicate the lysate in a cooled sonicator. This usually works for me.
Hi there,
As others have mentioned to you the viscosity that you get is due to the presence of too much DNA which willingly or unwillingly would bind lots of proteins or traps lots of proteins. So, your first priority is to get rid of your DNA which can be done by a simple salt and alcohol protocol. Then choose a solvent that could easily bring your protein in their native state with disulfide bonds intact back to life and then you can start your trapping of the protein of interest that you are looking for. Alternatively, you can sonicate to break up your DNA which might have deleterious effects on your proteins.
Good luck,
Chris
400 ul of Ripa for 1 well sounds quite high - did you mean 40 uL? If not, this volume might be contributing to your low protein concentration. Also, are you measuring the protein prior to running the gel? Weak bands could also reflect a transfer problem instead of a protein concentration problem.
You could try lysing with lower volume of ripa buffer (with protease and phosphatase inhibitors), incubating for 30 minutes on ice or rotating at +4 after scraping, sonicating samples briefly (on ice) after centrifuging (to shear the DNA), followed by further shearing with a 25 gauge needle and finally measuring the total protein.
Hi Diana, previously I used 200 µl but i faced the same problem of low protein concentration so I increased the lysis buffer volume. I am adding EDTA-free protease cocktail inhibitor but i dont have any phospahatase inhibitor with me right now. well, whether sonication should be done before centrifugation or after? what frequency should be used? i tried benzonase to reduce viscosity. Didnt work much
Dear Sonalika,
although RIPA buffer is quite often used in IP, it has the big disadvantage that the SDS and the desoxycholate contained in most variants of it break the nuclear envelope and thus liberate DNA. Of course you could try to get rid of the released DNA as my colleagues suggested. However, there is also another way to go: you might try to lyse the cells in a small volume of a buffer with physiological ion concentration (like PBS or TBS) plus 1 % Triton X-100. Under these conditions the nuclear envelope does not break (the membranes are gone, of course, but the nuclear lamins stay in place!) and you can spin the nuclei out easily. Try to keep the volume low to increase overall protein concentration. We are using 10 to 30 million of cells (lymphocytes) per ml, so you might consider scaling up your prep a bit. Lateron, e.g. in washing, you might use RIPA buffer to increase selectivity, but be careful, you might break potentially interesting protein-protein interactions! Good luck!
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