pavis Posted January 8, 2012 Posted January 8, 2012 Hi ! I am working with isolation of plant histone proteins. 1) Currently I am using acid extraction method to isolate histones from 10 g of leaves. 2) Bradford protein measurement to measure protein concentration in isolated samples, so that i can load desired amount of protein when I use western blotting. Problem is: when I run all the Histone samples on a gel to perform western blot, protein bands migrate at different pace and stop at different positions(first band at ~15 KD, second and third protein band ~20 ). even though all the bands contain same sample Why I worry so much because I use specific antibody, they should be located at one particular molecular weight. can some one help me with this Regards, Pavis.
CharonY Posted January 9, 2012 Posted January 9, 2012 Do you mean that you get different migration in the same gel when loading the same sample into different pockets, or do you get multiple bands in the same lane, or are they different samples (or extractions) in the same gel are are they different blots?
pavis Posted January 10, 2012 Author Posted January 10, 2012 (edited) Do you mean that you get different migration in the same gel when loading the same sample into different pockets, or do you get multiple bands in the same lane, or are they different samples (or extractions) in the same gel are are they different blots? Hii No, I dont have multiple bands in same lane. I get bands at different positions in replicates on the same gel. For instance, I have my control in first lane and First acid extraction sample in second lane, and second extraction third lane. When I run the gel and developed blot against specific antibody, I found one band in each lane at different positions. see picture below Edited January 10, 2012 by pavis
CharonY Posted January 10, 2012 Posted January 10, 2012 It is hard to assess from that blot, but it could be minute run differences (as e.g. a smile). To me it seems that the run is slightly irregular and overloaded. I would repeat the experiment with less protein (so that one gets a nice band instead of a blob) and put the control/marker left and right to the sample. That way you can see whether the gel ran straight.
pavis Posted January 10, 2012 Author Posted January 10, 2012 It is hard to assess from that blot, but it could be minute run differences (as e.g. a smile). To me it seems that the run is slightly irregular and overloaded. I would repeat the experiment with less protein (so that one gets a nice band instead of a blob) and put the control/marker left and right to the sample. That way you can see whether the gel ran straight. Okay, I would go with that suggestion and load less protein and check once again. Thank you It is hard to assess from that blot, but it could be minute run differences (as e.g. a smile). To me it seems that the run is slightly irregular and overloaded. I would repeat the experiment with less protein (so that one gets a nice band instead of a blob) and put the control/marker left and right to the sample. That way you can see whether the gel ran straight. Okay, I would go with that suggestion and load less protein and check once again. Thank you
CharonY Posted January 10, 2012 Posted January 10, 2012 (edited) I should also mention that depending on the precise procedure DNA could form complexes with histones and thus change the migration behavior somewhat. Edited January 10, 2012 by CharonY
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