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western blotting troubleshooting

westernblot smallprotein gelstaining

Best Answer mdfenko, 09 November 2018 - 01:21 PM

or is that the stained gel after transfer?

 

it looks to me like:

 

the gel is too high a percentage acrylamide/bisacrylamide (either or both)

 

too much salt in the sample

 

potassium salt in the sample (causes sds to precipitate as kds)

 

old cloner is correct in saying that you have given no information

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#1 Vui

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Posted 08 November 2018 - 06:51 PM

Please someone give me any advice sad.png

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#2 OldCloner

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Posted 09 November 2018 - 05:52 AM

So this is a Coomassie stain, not a blot...Well, something went wrong with your electrophoresis, but since you haven't told us what you did, or whether this is a new problem, or the first gel you've ever run, we can't even guess how to help.  Have you run these gels successfully before? If so, what did you do differently? My wild guess is a buffer or gel formulation problem.


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#3 mdfenko

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Posted 09 November 2018 - 01:21 PM   Best Answer

or is that the stained gel after transfer?

 

it looks to me like:

 

the gel is too high a percentage acrylamide/bisacrylamide (either or both)

 

too much salt in the sample

 

potassium salt in the sample (causes sds to precipitate as kds)

 

old cloner is correct in saying that you have given no information


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talent does what it can
genius does what it must
i do what i get paid to do

#4 Vui

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Posted 12 November 2018 - 05:40 PM

Thank you for your comment anyway (@mdfenko and OldCloner)

Actually I am new in this experiment. this photo is stained gel after loading the protein.

I did in 20% gel with an old buffer that It was prepared 6 month ago.

So yesterday I tried again with 15% gel with new protein extraction and new buffer the stained gel looked better for me.

I just want to updated the result for you.

Thank you for your helping again.smile.pngsmile.pngsmile.png

 

PS: my protein of interest is 15 kDa and I use 30 microgram per well. I would do the transfer today; hope it will be going well ^^

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  • KakaoTalk_20181112_193347971.jpg

Edited by Vui, 12 November 2018 - 05:56 PM.





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