Lately I ve been having a problem with electrophoresis of western blotting, I run 120v for 2h, i got strange band like picture. I used fresh buffer.
Please describe in more detail your experiment (picture to)? What is it ?, sds-PAGE or WB?
Can you visualize the bands in a SDS-PAGE? Do you have access to purified samples of proteins to use as positive control ?
Without additional information will be difficult to help you
What is strange about these bands? Are they at the wrong size or in the wrong sample?
Or do you just "don't like" their form? They look a little bit odd, because some are broader than others and some smear a bit. This often stems from the SDS-PAGE and not from the blotting procedure. I would try to lower the voltage for SDS-PAGE and check the gel and running buffers. You may also have debris left from the tissue, which can result in smearing. Try to spin down the samples prior to gel loading or optimize heat incubation. Different band width can stem from huge differences in total protein amount in samples next to each other. Higher concentrated samples can broaden and thereby narrow the lines next to them.
Band 1 and 5 look like transfer, antibody incubation or visualisation was not perfect as one side is darker than the other. There are many possible sources of error for this e.g. not enough pressure on the sandwich of membrane and gel, too low volumes of antibody or detection solution, awry incubation container for incubation with antibody or detection solution...
Hope any of this helps.
Add a bit of TE-2x to your DAB andAb solution. You will have best western blot results.
Hi Jamil can you send me the protocol preparing and using TE-2x, thanks
Are you using precast gels or gradient gels? If precast and you are talking about the weird shaped bands, this could be an issue with your stacking gel. Try to run the gel a bit slower (80V) whilst its compacting through the stacking gel, then you can run it faster as it all enters the resolving gel.
HI
Use an affinity-purified primary antibody
Optimize primary antibody concentration
Try another antibody
Check antibody specificity with blocking peptide,
Repeat immunodetection with secondary antibody alone to check for non-specific binding
Check and optimize gel electrophoresis conditions
Run gel at 4°C
hope this helps
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