for 2D gel electrophoresis i precipitate protein by chilled acetone at -20'C overnight. does it affect protein property for 2D experiment.
Hi Jayat,
protein extraction protocol and any protein solubilization protocol affects the composition of the proteome. in your case, some proteins may precipitate irreversibly in acetone.
Hi Jayant
Acetone in 2D gel is used in order to precipitate the total (whole) protein in the sample, this will concentrate the sample, reduce salts & improve the background of spots you'll get on SDS gel, I assume that you are not looking for activity thus this step should be OK even if the organic solvent (acetone) may denature the protein.
Hope this helps
Think about it. The nature of acetone precipitation is extraction of water from the structure. So.. the number of exposed functional groups can be changed. For a hydrophobic protein this might be no problem. For soluble proteins it could result in some problems. There is no substitute for experiment..try different methods and see.
my purpose is to identify protein spots by Mass Spectrometry, i am not looking for its activity
Jayant.. I was merely advising some caution. Acetone treatment may ( by dehydration) expose some functional groups which could change mobility. This might not be a problem but you should be cautious since you might not be able to compare the migration patterns with untreated samples.
Jayant I used acetone for participation of proteins and I test these proteins with SDS-PAGE and normal PAGE and there were not effected by acetone .Beside that I used it for precipitation of enzymes like peroxidase from plant and acetone did not effect the enzyme activity .But the must important thing is to keep the temperatures of acetone and the row materiel you use very low(-18 ) because high or normal temperatures can cause hydrophobic denaturation of proteins .
Hi Jasim,
SDS-PAGE is not a good method to determine success or failure of complex proteome extraction protocols. cells contain thousands of proteins, and SDS-PAGE simply doesn't have the capability to resolve them.
Hi David , I agree with you that SDS-PAGE is not a good method to determine success or failure of complex proteome extraction protocols, But Jayant question was( is acetone affect protein property for 2D experiment ) and if acetone not effect proteins properties in SDS-PAGE and normal PAGE(without SDS) and if enzyme like peroxidase did not lost its activity with acetone that is mean acetone is not effect of protein property for 2D experiment .
Hi Jasim,
Unfortunately, that's incorrect.
Some proteins will be lost in acetone precipitation and you will not observe them in 2D at all.
in addition, the fact that one enzyme may survive it without losing activity, doesn't necessarily mean other enzymes will fare similarly.
Hi David
If you read Jayant question again you will see that he already extract proteins and he was not asking about the the efficiency of using acetone for isolation of proteins , but his question was (( does acetone affect protein property for 2D experiment )) and my answer was (acetone is not effect of protein property for 2D experiment)
These are some paper how used acetone for preparation of proteins:.
1- Comparison of protein precipitation methods for sample preparation prior to proteomic analysis.Journal of Chromatography A Volume 1023, Issue 2, 16 January 2004, Pages 317–320
2-William J. Hurkman and Charlene K. Tanaka. Solubilization of Plant Membrane Proteins for Analysis by Two-Dimensional Gel Electrophoresis.Plant Physiology July 1986 vol. 81 no. 3 802-806
one last time:
Acetone precipitation will and does lead to the irreversible loss of certain proteins from a protein sample. other will not be recovered without the use of detergents. that means that you will see a subset (a large one) of the proteome and not the whole proteome.
Jasim could be right; for his purposes acetone treatment post-protein concentration might work. But be careful, even with pure protein samples dehydration with acetone or alcohols can result in significant conformation changes which can effect exposure of functional groups. Membrane proteins can be very changed. Just be cautious ,, run lots of controls.