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Western few questions


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#1 GFAP

GFAP

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Posted 16 July 2010 - 06:40 AM

Hi everyone!

I have a problem with my b-actin. I usually run my gel and see my protein and the loading control b-actin in the same nitrocellulose paper (I cut it). But now I have to see a specific protein at 40 kda, the same molecular weight of b-actin. How can I do? Do I have to strip my nitrocellulose and see b-actin? Or there is another method (another loading control?)

thanks

#2 krisztina

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Posted 16 July 2010 - 08:06 AM

Hi,

Both option can work.
You could use another loading control, such as
tubulin ~ 50 kDa (I use sigma t6199, mouse)
p85 (phosphoinositide-3-kinase, regulatory subunit 1), ~ 85kDa
COX-IV (~20 kDa) - mitochondrial, only on whole cell lysates (on a 10% gel it might be too small to be able to detect)

GAPDH (37 kDa), TATA-binding protein (37 kDa, only if you have whole cell lysate) are not so good for you, since you have a protein very similar in size)

Or you could use any other protein that is not effected by your treatment. Bear in mind that the protein has to separate well and has to be detectable on the % of gel you use for your protein of interest.
You don't necessarily need to cut the membrane, you can also re-probe your blot. If you have a different secondary ab for the loading, chances are even less that the loading control will be messed up by a re-probe.

The other option is to strip the membrane, which usually works fine with me (I normally do that). You can buy mild or more stringent stripping buffer (I use one from Pierce) or you can make up yourself (I used one with b-mercaptoEthOH added freshly, worked fine, but I've heard people using 0.1 N NaOH for stripping).

I would recommend you to do the actin last after all stripping as it usually is a very strong signal that is not that easy to strip off completely.
If you have concerns, you can stain your membrane with 2nd anitbody after stripping and see if you detect any leftover signal.

Hope this helps.
Krisztina

Hi everyone!

I have a problem with my b-actin. I usually run my gel and see my protein and the loading control b-actin in the same nitrocellulose paper (I cut it). But now I have to see a specific protein at 40 kda, the same molecular weight of b-actin. How can I do? Do I have to strip my nitrocellulose and see b-actin? Or there is another method (another loading control?)

thanks


Edited by krisztina, 16 July 2010 - 08:08 AM.





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