SDS and Bradford

[stefulj]

I have simultaneously isolated proteins, RNA and DNA from the same tissue sample, using TriFast (Peqlab). I have, according to the manual, dissolved protein pellet in 1%SDS. What would be the recommended method for quantification of the protein before PAGE and blotting? Does anyone know why (how) SDS (higher than 0.1%) interferes with the Bradford method?
Thanks,
Jasminka

[kiki06]

Hey Stefulj!

the best method is to quantify your protein after lysis. Although I think you did that. Just dont add SDS-loading dye yet I think you are ok.

SDS inteferes with bradford because it is a soap like molecule. It will interfere with the bradford reagent.

I think a high SDS will give you weird results not sure though...


cheers and welcome to the forum

[stefulj]

Thanks for welcome and fast response!

Tissue lysis is followed by chloroform extraction of RNA, ethanol precipitation of DNA, and then finally precipitation of proteins from the DNA supernatant (by isopropanol). I don't believe the protein recovery in the mentioned steps is 100%, so I can not rely on the quantification in the lysate.

After several washing steps you get the protein pellet that is very hard to dissolve (even in the SDS).

I am wondering would it be ok to simply measure A at 280nm, now when my protein solution, supposingly, does not contain other molecules that absorb at that wavelength??

Jasminka

[moleculardude]

Hello Jasminka,

I think measurement at 280 nm is ok to measure protein concentration, although in my experience it is usually just ok for an approximation if you dont have much protein and it is contaminated.

If you have a lot of protein and your washed pellet is quite clean, then it could be quite accurate. Try to use both 280 nm and a bradford, and see if you get results that are close to each other.

cheers, and welcome to the forum!

[kmunson779]

Maybe try the Lowry Method (which, as I understand it, tolerates and even uses SDS)?

Hey, here's one explanation for why the bradford gets messed up with SDS: "Excess detergent interacts hydrophobically with the dye and causes a wavelength shift and an overestimation of protein." (reference on the page: sbio.uct.ac.za/Sbio/documentation/Bradford%20assay.html)

[stefulj]

Hi!
Thanks to all for inputs!
I have performed measurement at 280nm, and obtained results in the expected range.
Jasminka

P.S.
For anybody interested, by putting "brad assay" into Google search, as the first match you get a nice overview of protein concentration assays (I am not allowed to post the URL until I send 15 posts or more...)

[milee]

Originally Posted by kmunson779

Maybe try the Lowry Method (which, as I understand it, tolerates and even uses SDS)?

Hey, here's one explanation for why the bradford gets messed up with SDS: "Excess detergent interacts hydrophobically with the dye and causes a wavelength shift and an overestimation of protein." (reference on the page: sbio.uct.ac.za/Sbio/documentation/Bradford%20assay.html)

will 0.1% triton X-100 will cause similar problems with Bradfod analysis