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  2. → Viewing Profile: Posts: mhr

mhr

Member Since 23 May 2012
Offline Last Active Mar 06 2013 09:55 AM
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Posts I've Made

In Topic: Problems with IPs

29 May 2012 - 08:12 AM

Any ideas?

In Topic: got band in control positive but not in sample...

25 May 2012 - 08:59 AM

Can you give more detail as to what your experiment is? What kind of samples are you running- vector expressin cells, endogenous lysates, animal tissues? What are your running conditions?

In Topic: SDS-PAGE gel and running buffer

24 May 2012 - 12:34 PM

I would recommend checking your transfer apparatus guide, or speaking with a rep- some machines will allow transfer 'stacking' in sandwiches (filter paper, gel, membrane, filter paper, gel, membrane, filter paper), but this often depends on a) your baseline transfer efficiency, and B) the specs of your machine.

In Topic: Trouble Western Blotting a 360kDa Protein

24 May 2012 - 12:31 PM

This may be a stupid suggestion- but if you are using nitrocellulose you may want to check your pore size. I know that the nitroceullose commonly used at our institution is pore size 0.45um, which allows proteins over about 110kDa to blow through during transfer (in my experience). Try a 0.2um pore size, or PVDF (I personally have much better results with PVDF for larger proteins).

In Topic: Signal stuck on top of seperating gel

23 May 2012 - 03:50 PM

Can you be a bit more clear by what you mean by "stuck"? Is the sample getting stuck while running (ie: the loading dye is not moving), or are you seeing your protein run higher when blotting? 10% seems a bit high for a protein that large, I would recommend a 7.5% with a 3-5% stacking. Alternatley, have you started using a new batch of SDS-loading buffer that may not be made properly and may not be denaturing as it should be? Have you run samples from these lysates previously? What protocol are you using?

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