Hi everyone,
I have developed this WB detection done with primary and secondary antibody conjugated with HRP. The expression of the protein on the right was done in auto inducing medium with E. coli. Do you know why the detection shows multiple bands? Could it be that the DNA interferes with the specificity of binding? I purified the protein from the mixture from which the analysed sample comes from and in the end a net band is visible con the Coomassie. Still I can't explain why there are multiple bands. The primary antibody is monoclonal and in IPTG induced fermentation one band only is observed, could it be protein degradation? But at higher mw?
All samples have been reduced with DTT 50mM final conc
cheers
Hi Saverio
I agree with Sebastian's comments above, and most likely the higher molecular bands are product of the aggregation of the induced protein. Alternatively, If you know your protein may be binding to DNA, then a defective sample buffer could also cause the higher molecular weigh bands because of the interaction of the protein with DNA at different stechiometric rates. You could treat your sample with DNAses before denaturing your sample to see if that could be the case.
In any case make sure you use fresh buffers.
Cheers,
hi, thank you for the reply, the description of thei mage is not very clear I understand. The lane in the middle with sharp bands is the ladder while lanes on the right of the marker are: 2 filtrated samples retentate on 300 kd and then 2 constructs autoindued. The antibody looks pretty much specific when there is no DNA in the sample. I tried to add 250 U/mL of Benzonase but i still have DNA leftover dragging proteins and that Benzonase is pretty much expensive!
Did you check that the pH is around 7-8 in your sample when you add the Benzonase? How long did you treat it?
If you can get DNAse it will be a cheaper option although you'll only digest dsDNA, but i don't think this would be a problem here.
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