[pavis]

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]

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]

CharonY, on 9 January 2012 - 06:19 PM, said:

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

Attached thumbnail(s)

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This post has been edited by pavis: 10 January 2012 - 03:22 PM

[CharonY]

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]

CharonY, on 10 January 2012 - 06:34 PM, said:

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, on 10 January 2012 - 06:34 PM, said:

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]

I should also mention that depending on the precise procedure DNA could form complexes with histones and thus change the migration behavior somewhat.

This post has been edited by CharonY: 10 January 2012 - 08:14 PM