Hi Anand,

Try this thread: https://www.researchgate.net/post/How_do_you_prepare_adipose_tissue_for_an_ELISA

Should yield protein good enough for western blots too.

Lipids interfere with the BCA assay:

http://www.sciencedirect.com/science/article/pii/0003269786903180

This interference can be prevented by including SDS:

https://www.ncbi.nlm.nih.gov/pubmed/1443532

Thank you Murtaza and Adam!

I want to ask another question on this thread, I'm doing a BCA on HT-1080 cells treated with specific fatty acids. I performed TLC on a subset of samples to get the lipid content of specific phospholipids, and now I need to perform a BCA (or maybe Qubit, if BCA turns out to be complicated) on the other subset to normalize the lipids to the amount of protein in the sample (for turn over of LPCATs, etc.).

From what I read from the abstract of the paper, "This apparent lipid effect was eliminated by the addition of 2% sodium dodecyl sulfate to samples prior to the analysis.", and the Thermo Fisher protocol says the same thing. So then my confusion is are you adding 2% SDS to your sample for a final concentration of ________% (which is not helpful), or add SDS to a final 2%. I'm mostly asking because I can't get access to the paper, and the assay manual is just as convoluted.

I need to get these samples processed soon, and don't really do a lot of protein work, so this needs to be validated quickly to run my samples. Thanks for any help.

I think the intention is for the final SDS concentration in the samples to be analyzed to be 2%. Since adding the SDS from a stock solution (typically, 10% w/v) slightly dilutes the samples, you should account for this dilution after the protein measurement is made.

For example, if you add 25 µl of 10% SDS to a 100 µl sample to get 2% SDS, the sample has been diluted to 4/5 of its original concentration. Therefore, after the assay is done, you should multiply the protein concentration in the original samples by 5/4. However...

The standards used for the BCA assay (usually bovine serum albumin) should contain the same amount of SDS as the samples. If you treat them the same way as the samples, that is by adding the SDS to them after they have been prepared at a range of concentrations, then you don't have to do the correction stated above, because it will have been incorporated already into the standards.