unsoluble precipitated proteins

5 posts / 0 new
Last post
ettore
ettore's picture
unsoluble precipitated proteins

Hi all!
I need to precipitate protein extracts in order to perform a derivatization reaction of carbonyl groups with 2,4-DNPH. According to the old original protocol everyone is referring to, protein extracts are precipitated with TCA and resuspended in 2N HCl /10mM 2,4-DNPH.
My troubles: After TCA precipitation I can by no means resuspend proteins in HCl/DNPH, absolute plastic! I tried with a different precipitation (Ethanol/Methanol/Acetone 50-25-25) but I had more plastic..
Question 1: Maybe the problem is not the precipitation itself but resuspending in HCl?
Question 2: does anyone know what is 2N HCl for in the derivatization reaction (Brady's test)? Do I need it?
Question 3: The same reaction in small scale is easily performed with the oxyblot kit in fc 3% SDS. Should I try to resuspend pellets in 3% SDS adding the HCl/DNPH mix in a second time?
Thanks for help and suggestions
ettore

g a
g a's picture
Hi Ettore

Hi Ettore

I probably can help you out with the solubilization problem.

What temperature are you carrying out the precipitation? Try and maintain as lo temperature as you can. Most of the times the insoluble protein is the protein that is denatured.

If you have nothing to do with the functional activity of the protein, then solubilize your sample in 2M thiourea +7M Urea or alternatively in 1%SDS prepared in HEPES ph 7.4.

After TCA precipitation, a quick wash with 2N NaOH of the pellet also helps in improved solubility of the proteins there after.

Pellets may take longer durations for solubilization, homogenize the pellet and then add either of the above mentioned solutions for quick solubilization........because of the increased surface area.

ettore
ettore's picture
 Hi Argerine,

 Hi Argerine,
thanks for your suggestions..
I precipitate the proteins at -80° and centrifuge the tubes at 4°.
Solubilizing the proteins in SDS/Hepes is something to try, and rinsing the pellet with NaOH is also a possibility. Still I don't know if it is so important to derivatize proteins in an acidic environment...I hope someone out there has the answer..
Ettore

Sami Tuomivaara
Sami Tuomivaara's picture
ettore,

ettore,

The carbonyl group derivatization by DNPH requires acidic environment. You can look up the mechanism by googling Brady's test mechanism.

I've always done my TCA precipitation at -20 or on ice... I've never heard anyone doing it at -80. Maybe the combined effect of TCA and -80C will cold shock the proteins into a pellet that is very hard to redissolve... Just a guess.

Maybe the precipitated protein would be easier to redissolve in HCl without the derivative. You can try to redissolve the protein in just HCl and later add DNPH stock in HCl.

Cheers,

scraggyterrier
scraggyterrier's picture
The DNPH protein carbonyl

The DNPH protein carbonyl assay is badly described in many papers. I agree with you that very few proteins will resuspend well in 2 M HCl (aq).

You can find a good protocol that works in:
Levine et al., "Carbonyl Assays for Determination of Oxidatively Modified Proteins," Methods in Enzymology 1994, 233, 346-357

This paper focuses on using HPLC to separate unreacted from bound DNPH, but it's easy to modify to work with just a spectrophotometer.

The way I do the assay is to resuspend the protein pellet in 500 µL 6 M urea in PBS. I then add 500 µL 10 mM DNPH in 2 M HCl (aq). The HCl overcomes the PBS to provide the acidic environment required for the reaction, but everything stays in solution. After a few minutes incubation I precipitate by adding 100% (w/v) TCA (aq) to ~10%, followed by washing the pellet with 50/50 ethanol/ethyl acetate until the washings no longer appear yellow. Pellets can then be resuspended in 6 M urea in PBS and absorbance at 276 nm and 370 nm measured. Molar absorptivity coefficients at these wavelengths for bound DNPH are given in the paper as 9460 and 22000, respectively.

Hope this is useful,

Dave