I would like to study proteins expression but now i am in try condition process.
I used PBMC for cell lysate preparation and the protein concentration always had negative value (-1 to -2). Anyway I have tried to run the SDS-PAGE as well as blotting and there has shown a specific band.
The problem is negative value, I can not adjust protein concentration for the same quantity in each batch and the quality of experiment.
I already tried 1 to 8 million cells per mL. I measured protein concentration via both nanodrop and bradford method. So I do not think that the neg value is come from measurement method, because both methods got neg value.
This is my lysis buffer recipe; 20mM Tris base, NaCl, EDTA, Triton-X and SDS. I used this lysis to prepare other cell types and they have positive value, so I am not sure it because of lysis buffer. The most people use RIPA buffer but my lab do not have some chemical to make RIPA buffer (NP40, sodium deoxycholate).
The question is: Where is the problem of my cell lysate and what should I do next??
For the figure explanation: I used only anti-beta actin (band size shown in lower band in lane 2-3). I do not know what is the upper band of Lane 1 but I used PMA activated PBMC for cell lysate preparation while lane 2 and 3 are viral infected PBMC and PBMC lysate, respectively.
Hi!
I think the problem is that you used Bradford-assay but your lysis buffer contains SDS. "When SDS concentrations are below critical micelle concentration (known as CMC, 0.00333%W/V to 0.0667%) in a Coomassie dye solution, the detergent tends to bind strongly with the protein, inhibiting the protein binding sites for the dye reagent. This can cause underestimations of protein concentration in solution."
There are some other methods (like the BCA-kit) which allows for quantification of your samples even with SDS. I used that with the same lysis buffer as yours, and it works just fine.
I hope this helps!
I agree, you used an inadequate method for determination of protein conc. Bradford detects not only proteins, it also reacts with detergences e.g. in RIPA.
Since your "blank" also contains these detergences - and your protein amount is possibly not that high - the difference between "sample" and "blank" is neglectable which may result in low measured (maybe negative) amounts, which are in any case physially not possible.
If you use detergence-containing lysis buffers you should use a more adequate method (e.g. following Lowry) to get a correct protein concentration value.
However, you picture clearly shows that there is protein (cells) in your sample...this is absolutely correct.
I would also recommend to do BCA assay/Smith assay instead, as this is compatible with the buffer containing detergents.
- Badges
- Science method