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benjamin 09-13-2007 02:48 AM

Plant Protein Extraction Mass Spec Analysis
 
Helo everyone!
i have a problem in extracting plant proteins for mass spec analysis:wacko:. any suggestions to this? thanks a lot!

admin 09-13-2007 08:21 AM

Re: Plant Protein Extraction Mass Spec Analysis
 
Hello there Benjamin,
could you please explain what the problem is exactly?

thanks!
admin

danfive 09-13-2007 05:24 PM

Re: Plant Protein Extraction Mass Spec Analysis
 
What is your extraction method? Are you running gels, digesting? etc...

benjamin 09-17-2007 08:44 AM

Re: Plant Protein Extraction Mass Spec Analysis
 
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 09-17-2007 03:46 PM

Re: Plant Protein Extraction Mass Spec Analysis
 
Quote:

Originally Posted by benjamin (Post 4875)

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

Try this troubleshooting guide --> [Only registered and activated users can see links. Click Here To Register...]

Quote:

Originally Posted by benjamin (Post 4875)
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.

Quote:

Originally Posted by benjamin (Post 4875)
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 10-09-2007 04:55 PM

Re: Plant Protein Extraction Mass Spec Analysis
 
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 --> [Only registered and activated users can see links. Click Here To Register...]


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