Hi
I am doing western blot. After following the full length protocol, i examined the membrane on Biorad analyzer. The bands of my interested protein have poor visiblilty (14kDa). In addition, these bands are not visible at low resoultion. But when i increased resoultion the backgroung becomes more black. i have also attached the figure (arrow indication protein of interest). is these results are acceptable? furthermore, is there anyway to clear the background and gain more visibility of the bands of protein of interest?
kindly give some valuable suggestions.
you could also try using a tris-tricine western blot which separates lower molecular proteins better. It seems that you are not really concerned with anything except the 14 kDa band. This system separates the higher molecular weight proteins, which appear a lot higher in the gel with less separation, from lower molecular weight proteins, which appear and separate for the majority of the gel.
you can also try by increasing washing duration. 15-20min 3 times. I get very clear background.
May be you can try adding 5% BSA as a blocking solution in TBST. Increase the desired protein concentration
Expose manually in biorad gel doc..
Adjust the image background accordingly.
Hope that helps you.
First, try the suggestions given by others...
If works, it will be fine.....
Or, load a negative control if negative control will show no band as like as your target band, then I think you can use it for publication.
Anyway, it will depend on reviewers and editor
You can also play with the antibody concentrations, in particular the secondary. I'd perform PAGE and transfer with all lanes containing the same sample, cut the membrane in strips (after Ponceau staining) and then use a serial dilution of antibody to incubate the strips.
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