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Electrophoretic Mobility Shift Assay Forum EMSA Forum. Discuss the Electrophoretic Mobility Shift Assay protocols or technique and the assessment of DNA and RNA binding proteins here. Also post questions and troubleshooting about the shift assay.


EMSA problem

EMSA problem - Electrophoretic Mobility Shift Assay Forum

EMSA problem - EMSA Forum. Discuss the Electrophoretic Mobility Shift Assay protocols or technique and the assessment of DNA and RNA binding proteins here. Also post questions and troubleshooting about the shift assay.


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  #1  
Old 11-04-2008, 12:40 AM
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Default EMSA problem



Hello

I have performed EMSA and could see the shift after SYBR green staining.... but unforunately I could not see the shift for the same sample when I am using the radiolabelled probe.

only difference i could sense is the DNA conc. , in radiolabelled probe ( DNA conc. very less) compare to DNA what I have used for the SYBR green staining ....

why I am not observing the shift when i use radiolabelled probe !

please give me your suggestions...

thanks in advance and waiting for ur suggestions...
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Old 11-04-2008, 03:46 AM
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Default Re: EMSA problem

Hi Rmohan,
i can understand your frustrations. What probe did you use: P-32 or other? What nucleotide did you label? Is the probe made the exact same way in both experiments (salt, buffer, conditions)? Have you tried non-radiolabeled probe?
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Old 11-04-2008, 12:08 PM
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Default Re: EMSA problem

hello

thanks

I have labelled the probe ( PCR fragment ) with end labelling (T4 kinase- gamma P32) and purified.

Iam sure probe is fine on gel with intact band , same PCR fragment used for the experiment and stain with ETBR or SYBR I could observed the shift , but not in the radiolabelled probe !... same conditions for the both the expts , no change at all.

I have not tried with non radiolabelled probe ( I mean not DIG labeling).

lookinig forward for the suggestions....
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Old 11-05-2008, 01:11 PM
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Default Re: EMSA problem

hi rmohan,
can you post you image?
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