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Plant Protein Extraction Mass Spec Analysis

Plant Protein Extraction Mass Spec Analysis - Proteomics Forum

Plant Protein Extraction Mass Spec Analysis - Post Questions and Discuss Proteomics, Proteomic Bioinformatics, Proteomic Techniques such as 2-D, Mass Spec etc.


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Old 09-13-2007, 02:48 AM
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Talking Plant Protein Extraction Mass Spec Analysis



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

Last edited by admin; 09-13-2007 at 08:05 AM.
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Old 09-13-2007, 08:21 AM
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Default Re: Plant Protein Extraction Mass Spec Analysis

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

thanks!
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Old 09-13-2007, 05:24 PM
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Default Re: Plant Protein Extraction Mass Spec Analysis

What is your extraction method? Are you running gels, digesting? etc...
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Old 09-17-2007, 08:44 AM
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Unhappy 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.
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Old 09-17-2007, 03:46 PM
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Default Re: Plant Protein Extraction Mass Spec Analysis

Quote:
Originally Posted by benjamin View Post

Problems;
Not sure why i couldnt get the proteins focussed in the first dimension (IEF).
Try this troubleshooting guide --> [Only registered users see links. ]

Quote:
Originally Posted by benjamin View Post
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.

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Originally Posted by benjamin View Post
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.

Last edited by danfive; 04-25-2008 at 05:11 PM.
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Old 10-09-2007, 04:55 PM
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Default 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.

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