I added it after grinding the tissues in homogenizer. Will it affect the protein extraction process because my protein of interest is missing from the samples.
You may want to clarify what you mean.
A common workflow for tissue extraction involves chilling the sample on LN2, then pulverizing it with LN2 chilled equipment (e.g. a mortar and pestle), then adding an appropriate volume of lysis buffer to an aliquot of the powder. The lysis buffer added at this step generally contains some detergent, but it is technically possible to extract soluble proteins in the absence of such detergents.
The way you've phrased the question almost makes it sound like you added lysis buffer *before* pulverizing the sample. That would be an odd workflow, but in theory it could still work.
If the buffer you added during pulverization lacked any detergent, you could in theory add it upon thawing the sample. Detergents are only effective in the presence of liquid water, which would not be present in an LN2 chilled sample/buffer mixture.
Thanks Dr. Bower for the reply. I generally put the tissues on Dry ice and then in - 80 C. These tissues that I talked about are came from - 80C then I add lysis buffer without detergent and then I homogenize using homogenizer for 30 seconds and then only detergent is added. Do you think there is an issue?
Raussie Vaid The solution to this problem will be protein-dependent. If you've never extracted this specific protein before, I'd first do some reading about what other people have done (if it's been done before.) Then, do a screen using different extraction buffers to see which condition will give you the most soluble protein. Some conditions you could test out are different detergents (CHAPS, Triton-X), different pHs (you want to stay away from the pI of your protein of interest), and different concentrations of salt. I've seen some proteins that love almost no salt and some that love 1M salt. It all depends on your protein, so it really has to be determined experimentally.
Another option is to do a 2-step extraction, where you resuspend the insoluble pellet from your first extraction in a different buffer to try and solubilize the protein.
Lindsey's right. Your extraction may have worked, but you'd need to determine that empirically (e.g. run your assay). Adding detergents post-homogenization is odd, and it means that they will not have contributed to the extraction of the proteins at all - it would only help stabilize the proteins in solution after the extraction.
If it wasn't too hard I'd run the assay and then decide whether I needed to redo the extraction, assuming I still had enough powder left to do another extraction.
Another option to test your protein quantity more quickly, albeit qualitatively, is to get an antibody against your protein and run a western. Just make sure you load the same amount of lysate in each lane (which will probably be a very small amount because of all the different stuff in there.) ...This is assuming you can quickly run a western and can afford to buy a monoclonal antibody. I'm in industry, so I have the Thermo iBlot kit and I can run a western in less than a day. I realize not every lab has those resources!
Thank you for the suggestions. I already ran the western and didn't see the band which was suppose to be there. Some samples have protein of interest in them but in some samples they don't have.That why I am really concern what to do?
What is included in your lysis buffer?
What kind of homogenizer do you use?
I agree that adding the detergent afterwards is not really helpful. Where is your protein located in the cell? Maybe you need the detergent in your lysis buffer to access the protein.
Raussie, if your assay detected nothing in the samples, and if you included appropriate controls to ensure that it could detect anything at al (e.g. blotting a sample from separate extraction), then I'd say it's time to re-extract the samples with a more-conventional extraction procedure (e.g. into buffered 2% SDS or RIPA). Your colleagues may have extraction procedures that they could share.
Hi all, thank you for all the comments. I used both Ripa and He's buffer but without detergent in it and added in after homogenizer. I repeated the extraction and after detergent after but I had to votex the samples vigorously and now I could see the band in western blot. But still if adding detergent later is causing this, then I will add detergent while homogenizing. Protein name is RIP3.
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