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2D Blots problems


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#1 biochem06

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Posted 20 July 2016 - 01:30 AM

Hi,

 

I'm running 2D gels then transferring to PVDF membrane. Up until now no problems.

 

This recent batch however I'm having issues with.

The first issue the ladder transferred perfectly but no protein transferred to membrane (there was 200 ug protein total on the 2D gel). Why would the ladder transfer and not the proteins? Ponceau on the blot revealed no spots and when I coomassie stain the gel there are some spots still on the gel but not many, suggesting they should have transferred.

 

The second issue is this weird horizontal line in the blot when I develop it (see attached images). It's not a "streak" from the 2D gel. It's something else that I haven't seen before and its not appearing in the coomassie stained gel - just the transferred blot. What could it be?

Attached Thumbnails

  • Picture2.jpg


#2 mdfenko

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Posted 25 July 2016 - 04:46 AM

is this a new lot of membrane?

 

if so, is it the same pore size as the previous lot?

 

is it older than the previous?

 

did you use fresh transfer buffer?

 

are you sure you have even contact between the membrane and gel?

 

did you presoak the gel in transfer buffer?

 

a little more information would be useful.


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#3 biochem06

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Posted 26 July 2016 - 08:24 AM

Hi

 

This is actually a new lot of membrane and I do think it is faulty. The transfer buffer was fresh. I wouldn't say I presoaked the gel in transfer buffer - maybe just for 30 seconds. maybe I need to leave it in the transfer buffer for 15 minutes or so to allow it to equilibrate.

 

I developed another blot and had problems with it too. There were no reactive spots but the whole image and screen had a "speckled" appearance. Quite similar to the fuzzy/speckling seen in the image I have attached (I have borrowed this image it's not mine). What would cause this speckling/fuzzy effect over the whole screen (not just the area of the blot)?

 

fuzzy blot.jpg



#4 mdfenko

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Posted 27 July 2016 - 10:07 AM

one thing that can cause that type of background is the texture of the blotting paper, especially if contact with the gel is not uniform.


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#5 biochem06

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Posted 01 August 2016 - 12:38 AM

Many thanks once again mdfenko smile.png



#6 mdfenko

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Posted 02 August 2016 - 03:28 AM

one more thing, the filter papers used in the transfer may be a little too dry.

 

i'm attaching a book on western blotting (by emd millipore) which may help in troubleshooting.

Attached Files


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#7 biochem06

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Posted 16 August 2016 - 01:50 AM

Thanks mdfenko, appreciate it






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