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Western Blot Forum Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


Western blooot troubleshooting

Western blooot troubleshooting - Western Blot Forum

Western blooot troubleshooting - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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  #1  
Old 08-14-2006, 09:27 PM
Pipette Filler
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Default Western blot troubleshooting



Dear Friends
Ia m facing a problem with non specific reactivity with proteins in western blott and Iam gettting tooo much background for G6PDH antibody and I need importnat information on the solution used for antibody solutions
I am preapraing my primary antibody in 2 percent milk pwder is it right ?????
Shall I use PBS to prepare my primary and secondary antibody ?????
Please send rely soon
Vivek

Last edited by admin; 02-24-2007 at 03:56 AM.
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  #2  
Old 08-14-2006, 10:21 PM
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Default Re: Western blooot troubleshooting

Hey Vivek,

welcome.

5 % Blotto is usually standard in 1% TBST. I have never tried PBS although you should be ok.

I use 1% TBST for all antibody dilutions.
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Old 08-15-2006, 05:24 AM
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Default Re: Western blooot troubleshooting

Dear
Let me ask one question can we use PBS to dilute our primary and secondary antibodies rather than TBST and How can I solve the problem of waek signal for my G6PDH protein expression in heart.
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Old 08-15-2006, 10:41 PM
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Default Re: Western blooot troubleshooting

Dear Pintu

I would load more protein.... or even better, lyse the cells (whole amount) in a small amount of SDS-Page LOADING buffer.. ie 100 ul... this will show your protein for sure...

check out [Only registered users see links. ] they have some good articles on this... i think PBS is ok...
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