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Separating proteing with difference of 5Kd on western blot

Western Blot

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4 replies to this topic

#1 Preeti Dave

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Posted 18 December 2012 - 03:20 PM

Hello everyone,
I am kind of stuck here with two proteins with almost similar molecular weight and I want to do Western blot of them as it is a
requirement of the project. Can someone guide me?? the weight is 90kd and 95 kd.
Thanks,
Preeti


#2 Preeti Dave

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Posted 18 December 2012 - 03:43 PM

My question is How will I cut my membrane as I dont have big difference in protein weight. And one more problem is I have three proteins ranging from 100 to 90KD!!! please reply me. I saw some relly good reply on how to cut membrane !!

#3 bob1

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Posted 19 December 2012 - 12:05 PM

You shouldn't need to cut the membrane - a long enough gel running time at 6-7% will resolve and separate the similarly sized proteins quite well. You can also use different species primary antibodies to detect the proteins (e.g. mouse for the 90, rabbit for the 95 and goat for the 100) and then probe with appropriate secondary antibodies. Note that for this to work well, you will need to avoid secondaries that are raised in a species you are already using. For instance a lot of secondaries for mouse primary antibodies are raised in rabbits (rabbit anti-mouse), but if you are also probing using a rabbit primary, this will bind the rabbit anti-mouse secondary. You can also get directly conjugated primary antibodies with HRP or alkaline phosphatase on them, so no need for a secondary antibody.

#4 Preeti Dave

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Posted 19 December 2012 - 04:53 PM

Thanks for you reply,
I am not sure what you mean by to use 6-7% ? is it a gredient of 6-7% ?? But I have to study 4more proteins on the same blot which has various molecular weights.
556kDa, two proteins with 95kDA, 90kDa, 65kDa and 26kDa.
and one housekeeping protein as well!!
Do you think whitout cutting I can perform this?? I have all antibodies from cross species!! It is really complicated.
If you have any suggestion for me please do post it.
Thanks,
Preeti

#5 bob1

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Posted 20 December 2012 - 01:13 AM

A gel of 6 or 7 % for resolving proteins of about 100 -200 kDa, above this you will need to drop to 4 or 5%. 556 is huge, I am not sure that you will be able to resolve this well at all with a SDS PAGE gel. You won't be able to properly resolve this protein on the same gel as the smaller proteins.





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