Scientist Solutions - 2D PAGE of wet plant proteins a problem?

proteome Posted 2/1/2010 11:24:12 AM	I have been working on plant rhizome materials. I have noted with concern that wet materials always gives inferior gels when compared to gels from dry materials. This is in terms of streaking, spot numbers, spoy intensity. The situation is the same even for 1D gels. This is despite the fact that i use the same extraction protocol (phenol based) and treat the proteins similarly! Why is this? could it be that wet materials have more interfering compounds and proteases than dry materials? Any suggestions on how to deal with the problem?
ARGERINE Posted 2/1/2010 11:35:48 AM	Dear Proteome My first suggestion is to never use the raw material as such for 2DE. Pecipitate your proteins by using any specific protein extraction procedures like TCA-Acetone and then proceed for the down stream electroporesis. Although I haven't worked wit plant proteins but basic chemistry involved behind the 2DE remains the same and that is salts, carbohydrates, lipids and phenols interfere with the resolution. Just in case you are also getting streaking in the High Molecular weight region as well that may be due to presence of nucleic acids in your sample.
proteome Posted 2/2/2010 2:43:01 AM	Dear Argerine Thank you for your suggestions. I nomally extract my proteins using a phenol based method and precipitate proteins using 0.1M ammonium acetate in absolute methanol overnight at -20 C. The strange thing is that i have beutiful gels for dry plant samples but always get heavily streaked samples with wet plant samples. Dou you suggest that i use a different procedure for wet samples? Thank you again.
varsha Posted 2/2/2010 9:08:40 AM	I suspect that your extraction protocol does not remove lipids and/or DNA/RNA from protein samples. I have never run a 2D gel for plant samples but try to follow the protocol in this paper and see if it reduces streaking. www.sciencedirect.com/science http://jxb.oxfordjournals.org/cgi/content/full/57/7/1493 There is an older article for 2D with plant membrane proteins which may also give you some ideas. www.jstor.org/stable/4270049 You may just need to tweak your protocol just a little to get a better gel. Let us know how you get on. Good luck!
ARGERINE Posted 2/2/2010 11:09:41 AM	Hi Although Methanol should solubilize the lipids I recommend a nuclease treatment incase the streaking is only in the high MW region. Another point of consideration is If the proteins in your sample are completely solubilized or not? In case your sample is having denatured protein it would not be solubilized completely and the microprecipitate will give streaking. Centrifuge at 14000g or more for 30 min before loading and collect the clear supernatant and then only load. Try using TCA/ Acetone for your next 2DE.
proteome Posted 2/3/2010 2:15:20 AM	Thanks a lot for all your comments. I will make some modifications on my protocol and i will tell you what i get.
dineshibms Posted 3/3/2010 4:48:12 AM	Hi, i too got these problems while iam doing proteomics. If u follow cold acetone precipitation method it wil surely help in reducing the streaks. And also ur sample wil be free from lipids and phenols and also try to prepare all ur buffer mainly rehydration buffer freshly.. This is the procedure i followed ... Cool the required volume of acetone to -20^oc ↓ Place the protein sample (50 μl) in acetone compatible tube ↓ Add 4 times the sample volume of cold (-20^oc) acetone (200 μl) acetone to the tube ↓ Vortex the tube & incubate for 60 min at -20^oc ↓ Centrifuge for 10 min at 15000 g at 4^oc ↓ Decant & properly dispose the supernatant, protein sample should not to be disturbed ↓ Allow the acetone to evaporate at RT. Do not over dry the pellet, or it may not dissolve properly (~15-30 min) ↓ Resuspend in appropriate buffer (10 μl Rehydration buffer)
proteome Posted 3/3/2010 6:23:27 AM	Hie All I am very thankful to you all. I have modified my protocol and use a phenol protocol based on Hurkman and Tanaka (1986) with some modifications and also ammonium acetate precipitation. Among other changes, I have also resorted to centrifugation at 14000g for 30 mins before loading the supernatant. I have attached hereunder , some examples of gels i got from the old protocol and the new protocol. i will also use other protocols you guys have suggested for the sake of comparison. I am very greatful for your valued contributions. Please , i welcome all other advise and suggestions. Proteome
ARGERINE Posted 3/3/2010 11:37:55 AM	Hi From your electropherograms It seems there still are contaminants present in your final protein extract. Perform TCA acetone purification and solubilize your sample in minimum ionic strength buffer and Give atleast 1 cm stacking in your 1 dimensional gel and you will see the sharpe bands. If possible switch from glycine to Tricine PAGE.
proteome Posted 3/4/2010 5:26:13 AM	Thanks ARGERINE. I will try your advice and come back to you.