Plant Protein Extraction Mass Spec Analysis

[benjamin]

Helo everyone!
i have a problem in extracting plant proteins for mass spec analysis. any suggestions to this? thanks a lot!

[admin]

Hello there Benjamin,
could you please explain what the problem is exactly?

thanks!
admin

[danfive]

What is your extraction method? Are you running gels, digesting? etc...

[benjamin]

Hi admin and danfive,

thanks for replying. well, im trying to extract plant proteins to run in 2DE. However, i did not succeed in doing so and all that i got from the gel map was horizontal streaks (which suggest that the proteins werent even focused, i think). im also planning to analyze the proteins with maldi-tof once i manage to get proteins spots.

the protocols;
i grind the plant fruit mesocarps in liquid nitrogen and suspend it therafter in extraction buffer (0.1 M Tris-HCl pH 8, 0.5 M NaCl, 5 mM DTT, 0.2 mM PMSF). then i sonicate the mixture for bout 30 min before spin it down (14 000 rpm, 15 min). subsequently, i add TCA/acetone and incubate it at -20 o/n. i wash the precipitated proteins with acetone the next day and air-dry the pellet before resuspend in rehydration buffer for IPG strip.

Problems;
Not sure why i couldnt get the proteins focussed in the first dimension (IEF). is there any standard protocol to extract plant proteins for 2DE run and mass spec analysis? if i do extract the proteins in rehydration buffer, can i run the proteins in 1D PAGE?

thanks you so much. really appreciate any suggestion/comment.

[danfive]

Originally Posted by benjamin

Problems;
Not sure why i couldnt get the proteins focussed in the first dimension (IEF).

Try this troubleshooting guide --> http://www.chemsoc.org/ExemplarChem/...leshooting.htm

Originally Posted by benjamin

is there any standard protocol to extract plant proteins for 2DE run and mass spec analysis?

There should be; its been a long time since I worked with plants, but I remember (after grinding up the tissue) using a 5M KAc and high speed centrigation step to precipitate Starches? (or something), then transfering supernatant to clean tube, followed by high speed centrifugation, and collecting the supernatant for protein analysis.

Originally Posted by benjamin

if i do extract the proteins in rehydration buffer, can i run the proteins in 1D PAGE?

Can't say for sure, I assume your rehydration buffer is a 9M urea buffer.
I do know for certain that you can dissolve your protein pellet in 3M Urea, grind it gently to dissolve completely, then use it a Pierce BCA Microassay to determine your protein concentration accurately (BSA Stds & samples can be set-up/diluted in 3M Urea). This will also run normally on a 1D SDS-PAGE with the usual TGS buffer.

Remember you definitely want to quantitate protein, too much protein can cause your streaking horizontal and vertical, and mess up/slow down IPG focusing.

[danfive]

I just wanted to add this great article, "Reduction of proteins during sample preparation and two-dimensional gel electrophoresis of woody plant samples," as a reference for people interested in plant protein extraction and 2D gels, etc.

The link --> http://www3.interscience.wiley.com/c...95653/ABSTRACT