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Protein of 160 kda is present on 6% gel but gapdh housekeeping protein is not.


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#1 labrat640

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Posted 02 November 2011 - 02:51 AM

I have carried out a western blot for WRN, a protein of 160 kda. Protein was loaded on a 6% gel and the WRN band is present.
Bands for either GAPDH (approximately 40 kda) or tubulin (50kda) were absent after stripping the membrane and reprobing each time.

Is it because the protein sizes are too small for a 6% gel? A ladder is loaded and demonstrates different sizes on the gel (from 250 kda to 10 kda).

What loading control can I use?

#2 zienpiggie

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Posted 02 November 2011 - 08:09 AM

How long did you run it for? Does the 10 kda ladder remains in gel?

#3 labrat640

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Posted 02 November 2011 - 11:21 AM

I ran it for 30 minutes. Yes, the 10 kda ladder remains.
I have just seen a list of recommended gel concentrations, it suggests a 6% gel for 60-300 kda.
What control can I use for loading?

#4 zienpiggie

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Posted 02 November 2011 - 06:21 PM

Another question is, how long did you transfer it for? Are the antibodies new antibodies or has someone else tried them?

#5 labrat640

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Posted 03 November 2011 - 08:42 AM

20 V semi dry transfer for 1 hour. Antibodies were fresh.

The question is: can protein bands the size of GapDH and Actin be visible on a 6 % gel or not?

#6 mdfenko

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Posted 04 November 2011 - 07:08 AM

yes, they can, if you don't over run. it would be better if the gel was 7.5%.
talent does what it can
genius does what it must
i do what i get paid to do

#7 labrat640

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Posted 04 November 2011 - 09:07 AM

Thanks MDFenko,

Are there any loading controls larger than 60 kda?

I think the solution is to run a 7.5% gel. I will give it a try.




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