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Western blot with smeared bands


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3 replies to this topic

#1 lolifox

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Posted 24 July 2014 - 04:29 AM

Hi everybody,

I got a problem in my western blots which is really annoying me.

I dont know why I always obtain smear bands. (Picture from attached file)

May anyone help me, please?

Attached Thumbnails

  • Picture2.png

Edited by lolifox, 24 July 2014 - 04:29 AM.


#2 mdfenko

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Posted 24 July 2014 - 10:44 AM

a few possibilities (may be others and/or multiple causes):

 

too much protein

 

incompletely solubilized protein

 

aggregated protein

 

overdeveloped blot

 

antibody too concentrated


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

#3 lolifox

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Posted 24 July 2014 - 04:46 PM

Thanks you so much,

Attached files below is SDS PAGE. 

I think it's not too much protein loaded.

My protein is assembled protein.

Developed time: 1 min

Antibody concentration: 1st 1:5000 ; 2nd 1:7000

 

don't know the smeared bands come from.

Attached Thumbnails

  • SDS.png


#4 mdfenko

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Posted 25 July 2014 - 04:03 AM

remember that immunostaining is a lot more sensitive than coomassie (especially coomassie g). you could reduce the concentration of primary antibody.

 

you can try spinning out insoluble proteins prior to addition of sds sample buffer.

 

you might want to look into adjusting the denaturing conditions for your sample.

 

do you boil at 95-100C? if so, then how long? over boiling can cause aggregation, even in the presence of sds and 2-mercaptoethanol (or dtt).

 

you may want to try 60-70C for ~20 minutes.


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




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