hi im a beginner in lab work. i have been doing multiple runs of western blot to detect protein (be it protein of interest or housekeeping proteins) but i couldnt get a good band.
i realise recently that i had been skipping 1 step and that is after removal of media in cell culture, i forgot to wash it with PBS and the resulting cell lysate (after cell scraping and homogenization) has a pinkish color (remanants of media).
my question is could this mistake have resulted in the weak western blot signals? maybe there are protease in media or it could be that it overestimates the protein present in my lysate thus resulting in less than required lysate loaded into SDS.
EDIT: I did remove the media till its dry. so maybe there isint that substantial amount of media left but just enough to give a slight pinkish color in lysate.
1. If you don't remove serum containing medium by washing, it may interfere with your protein estimation and may reduce cellular protein conc. if you load fixed amount of protein. So, if I were you, I will load on volume basis (10ul or 20ul) and use house keeping proteins such as beta-tubulin, GAPDH etc as loading controls.
2. When you load cell lysates made without washing of medium, then you may see non-specific bands at 55kDa (heavy chain [HC] of IgG) and 25 kDa (light chain [HC] of IgG) due to cross-reactivity of IgG in FBS with your secondary antibody and may also mask band appearance of your protein of interest if it is close to HC or LC of IgG or BSA in molecular weight.
Yes what's left from the media could interfere with your protein dosage. The media has FBS and as you may know FBS contains different proteins and the BSA is the major one. So when you estimate your total proteins you are estimating also what lest as BSA in your sample. That's could explain why you having a week band. When you prepare a protein lysate you have to add a proteases and phosphatases inhibitors to preserve the integrity of your sample for any analysis you may need.
Good a luck.
1. If you don't remove serum containing medium by washing, it may interfere with your protein estimation and may reduce cellular protein conc. if you load fixed amount of protein. So, if I were you, I will load on volume basis (10ul or 20ul) and use house keeping proteins such as beta-tubulin, GAPDH etc as loading controls.
2. When you load cell lysates made without washing of medium, then you may see non-specific bands at 55kDa (heavy chain [HC] of IgG) and 25 kDa (light chain [HC] of IgG) due to cross-reactivity of IgG in FBS with your secondary antibody and may also mask band appearance of your protein of interest if it is close to HC or LC of IgG or BSA in molecular weight.
Hello Iwan
I had worked in western blot from lysate cells.
I think, more important is the use of inhibitors in your lysis buffer that the pink color.
To analysis the interference of the rest of culture medium with western blot results. You can try do a western blot with the same (approximately) medium volume with your antibody. In order to find interferences with your results.
Regards
Remainings of media could down the detection limit of your assay in the case of protein of your interest, but I dont think it is the only problem in your procedure. If you assume that the rest of the procedure was OK, the loading controls such as ACTB or GAPDH etc. should have given good band anyway. Consider including a positive control lysate to your assay or just any tissue homogenate which must result in a good band at least for the loading control.
Based on my experience, the most important thing is your lysis buffer and protease inhibitor cocktail. If you use the proper one in proper volume, seemingly there shoul be no problem
First, you have to aspirate cell culture medium then wash with ice cold PBS to remove any residual cell culture medium then add your lysis buffer (RIPA, SDS, etc) with protease and phosphates inhibitors . Any residues from cell culture medium will affect your protein concentration as it contains FBS.
Good luck!
If you removed nearly all of the medium, I doubt that is the source of your problem. I have had to do that on occasion and it has not led to no bands. There are so many other basic things that could lead to poor results though...
1) Are you seeing any bands or are you seeing nothing?
2) How many cells are you lysing and in what volume?
3) Are you using PVDF? If so, are you activating it with methanol?
4) Have you been using the same secondary for all of your blots? Do you know that it is working? Are you using it at an appropriate dilution?
5) Is you ECL or detection reagent working?
6) Do you know that your transfer is working? One easy way to tell is to stain your blot with Ponceau S after you take it out of the transfer apparatus. After washing with diH20 you should see protein bands on your membrane if the transfer worked. The Ponceau S will not affect downstream applications.
Good luck!
Hi,
did you solve the problem? I am having a similar one right now as I am using the same reagents and protocol as my colleague, but the proteins (checked by Ponceau) seem not to bind properly to the membrane. It improves a bit by transferring longer, but still not completely and it must be something in the samples, as other blots of mine, when I use other lysates work fine.
Any ideas?
hi aleksandra,
yes, in that mistake i forgot to wash and dilute cells with PBS after media removal. i washed twice with pbs before adding lysis buffer.
yes it turns out that my lysis buffer wasnt optimal. i replaced each and every constituents (about 10 or so?) that made up the lysis buffer. If you are a beginner in the lab, you might just make a mistake in preparing the constituents from scratch(adding wrong powder, or inappropriate pH). please dont forget to add protease/phosphatase inhibitors in your overall lysis buffer (pill commercialised type is the best)
if i recall, i also altered constituent of my transfer buffer. i added more methanol. 700ml miliQ, 200ml methanol, 100ml 10X transfer buffer/
Hm ok. Doesn't account in my case. I'm using the same buffers as my collegue, same inhibitors and washing the cells before lysis. Also the first blot came out well before I froze the lysates in -80 (what we always do and it never affected them).
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