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higher MW non specific band


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#1 Tfal

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Posted 15 May 2009 - 08:35 AM

I've been trying to Western blot a medium MW protein subunit (AMPKa1) for a little while.
My Westerns first showed a lot of unspecific bands. As I applied basic trouble shootingh advices (changing blocking agent, longer blocking, more or less 2nd AB, different gel %, etc) the general appearance of my blots got better: less non specific bands.
But one major problem remains, I get a very strong band at a higher MW (around 90-100 kDa), actually stronger than the targeted protein (63 kDa), for wich I do get a signal for my WT tissues , but faint...
The apperance of my higher MW non specific band is the same wether it's KO or WT samples (same gel and membrane)... so I gather it's not contaminants linked the sought subunit or a polymer comprising it.
I vaguely recall someone mentionning something about an artefact band at 90-100 kDa, but I can't retrieve the post...

Could (over) boiling be the cause? Would heating at 70 C help?
Should I try precipating my lysates with ammonium sulfate?

Thanks,
Tfal

#2 Aris

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Posted 15 May 2009 - 10:19 PM

I've been trying to Western blot a medium MW protein subunit (AMPKa1) for a little while.
My Westerns first showed a lot of unspecific bands. As I applied basic trouble shootingh advices (changing blocking agent, longer blocking, more or less 2nd AB, different gel %, etc) the general appearance of my blots got better: less non specific bands.
But one major problem remains, I get a very strong band at a higher MW (around 90-100 kDa), actually stronger than the targeted protein (63 kDa), for wich I do get a signal for my WT tissues , but faint...
The apperance of my higher MW non specific band is the same wether it's KO or WT samples (same gel and membrane)... so I gather it's not contaminants linked the sought subunit or a polymer comprising it.
I vaguely recall someone mentionning something about an artefact band at 90-100 kDa, but I can't retrieve the post...

Could (over) boiling be the cause? Would heating at 70 C help?
Should I try precipating my lysates with ammonium sulfate?

Thanks,
Tfal


call the company from which you got the I Ab and ask them if there is any known cross reaction with the Ab tha you are using. Companies usually know these things

#3 bob1

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Posted 18 May 2009 - 05:25 PM

I agree, cross reaction in antibodies is quite common.

Precipitation of the proteins won't work, presumably the cross reactive protein is in the lysate.

#4 Tfal

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Posted 19 May 2009 - 05:28 AM

well thanks for you imput... but I'm with a technician from the lab who provided us the AB. I did mention the recurring non specific band at about 90 to 100kDa. She had nothing to say about it and replied by giving general tips for reducing non-specific binding...
I would think that if it was a such a cross reaction, she would be aware of it.

Can someone tell me how ammonium sulfate precipitation helps for some situations (some WBs) and why it would be useless in this case...

Thanks,
Tfal.




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