I have no experience on that cells but I use Bradford assay to determine protein content. First y use WB lisis buffer or another lisis buffer for enzimatic determinations. YI also used another lisis buffer based on hiposmotic lisis. Once you have cell homogenate, make a BSA standadr curve as follows (BSA stock 1 mg/ml; stored at -20°C in 1 ml aliciotes) (0; 2.5; 5; 7.5; 10; 12.5; 15 ul in 100 ul final vol in destiled water)

Samples should be dituted to enter the curve. 

Once samples and standard curve is ready add 1 ml Bradford solution (40 mg comassie blue (G6250); 50 ml ROH 96°; 100 ml Phosfhoric acid 85%; final vol: 1L in destiles water). Better if use deionized water.

Incubate at RT for 30 min and read at 595 nm in 1-2 ml chambers.

You can adjust volum for ELISA too.

You cannot use the media with your secretome for protein estimation just like that. You will have to use either TCA (TriCarboxylic Acid) or Acetone to precipitate proteins from your media. Reconstitute this precipitated protein in your western blot lysis buffer and then proceed with protein estimation with BCA, lowry or Bradford method (whichever is convenient for you). I have always done it like this and it works. Let me know if you have any further questions.

https://tools.thermofisher.com/content/sfs/brochures/TR0068-Protein-assay-compatibility.pdf

Hi Euan,

The link shows a list of compounds and what concentration is required for compatibility with the BCA assay. You may be able to dilute specific compounds to meet the required concentration threshold (I'm unsure what your buffer is composed of). I've performed BCA assays on samples which contain high levels of DTT simply by diluting it to an appropriate concentration and these have worked out fine.

Hi Everyone,

So after being very silent for the past few weeks (I had to figure this out and give 2nd year tutorials), I have had some success.

Thanks Maria I found an interesting modified Bradford assay that seems to have worked (Linked below)

Thanks for the suggestion Avinash. Ideally that would be perfect but I'm also running a parallel study to identify precipitation methods which will aid MS/MS downstream so I wanted to determine the protein concentration prior to each precipitation method.

Thanks Laura. I've taken a look at similar lists before and unfortunately makeup of the chemically defined media we bought from Gibco is kept very secretive so we actually didn't have any reference to go on to what was actually in there. That being said there is definitely something in there that interferes with the BCA assay.

So to combat all of this I found some papers on secretome analysis alluded to the use of the Bradford assay.  I went one step further and used a modified Bradford assay (given below). I then further modified it to use the smallest amount of sample possible to reduce wastage (20ul) while keeping the Bradford concentration the same. The results were fairly promising and managed to detect the protein concentration in complex samples. We will only really know how accurate they were once I've completed the analysis on my protein precipitation experiments.

So in conclusion it looks as if the Bradford method can work if samples are diluted and the amount of Bradford reagent is reduced and measured at two different wavelengths (as described in the paper below). I'll keep everyone updated on my progress. 

Have a great Friday,

Cheers,

Euan

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3164080/

Hi Euan,

I have gone through the paper which you linked for the modified Bradford assay. I have a doubt regarding the unknown samples dilution. Can you share with me how much volume (ul) of unknown sample can be used for the dilution?

Thanks!