So I have been working on this Western Blot technique for my laboratory rotation and I still get uneven bands. In my last experiment the samples are arranged in the next way: 1 - 5 and once again 1 - 5. The problem I have in this particular case is that the bands between each duplicate are quite different.
That is, the band's magnitud between Sample 1 in the first duplicate and the second's duplicate are not the same. At first I thought it was a blotting problem, but the ladders are well blotted. So maybe the amount of protein that I'm using? or some kind of uneven antibody incubation?
Any kind of feedback is highly appreciated.
Thanks in advance!
Considering the ladders are present on both sides and only single bands are present in all lanes, there are a couple of reasons as to why your bands are not equally thick throughout:
The middle bands seem brighter than the outer, perhaps it is a transfer issue (uneven transfer).
It might be the concentration of protein was used less, may try different concentration first, even problem occurs, increase the volume of chemiluminescent reagent,
and also follow the answers given by the above persons.
Good lcuk
The transfer of protein ladder is not the best reference for stating a "good blotting" since it usually comprises highly soluble proteins in large amounts that can be easily transferred. My first impression was that there is a air bubble in the first lanes that interfered with transfer. You should check by reversible Ponceau S staining to see if this is the case. If you are sure there was no bubble, than maybe the membrane was not wetted/equilibrated properly at this spot. Particularly when using PVDF, make sure that your membrane is soaked in methanol and transfer buffer for about 5 minutes at least before setting up the transfer.
In some other cases, such kind of 'dark spots' can occur during blotting itself, if the transfer buffer at a distinct spot is consumed and membrane heats up. Usually, dry membranes do not bind antibody very well, resulting in uneven staining.
I am not sure if the picture represents the complete blotting membrane ? If yes, you should also make sure that your membrane matches the size of the gel to make transfer most efficient. Hope this helps, best rergards, Michael
Before western blot, I would quantify the total protein using BCA, or Bradford assay if your protein is pure. Then only load max of 40 ug of total protein per well.
Thanks to everybody who answered my post.
It seems it's a recurring topic with our transfer cell, as it has happened to my college, so I will try with another machine. Although I will definitely make sure there are no bubbles before the transfer, that there is enough chemoluminescent reagent and that the membrane is soaked in methanol (I use isopropanol) and transfer buffer 5 minutes at least before the transfer.
Also the membrane was cut 2 cm on the bottom so it could fit the containers we use to block and add the antibodies.
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