You better optimize electro-blotting time and voltage, membrane. Like for stable adsorption onto the membrane, pre-wet the membrane in methanol instead of water before transfer is recommended. For the size of your protein, PVDF of 0.45 uM is better. In addition follow the post-transfer steps carefully. Good luck

So with PVDF membranes you should make sure to activate them using methanol first, this means before you start the blotting, the membrane should be incubated in methanol for 15-30 seconds.

another important point is to avoid having any bubbles on the membrane, cause where there are bubbles, the protein will not transfer.

Hello less than 50kDa protein better to use 0.25 uM pore size methyl cellulose or PVDF. further membranes and gels need to be equilibrated with transfer buffer. hope this might be helpful.

Hello Sera,

If you only can't visualize one out of a few 30 kDa proteins in your Western Blot, I would start with checking the a/body specific for the protein such as:

use a fresh aliquot,

use a higher concentration and/or longer incubation time,

use a different manufacturer,

use a different antibody.

Also, check the compatibility of your visualization method with the primary a/body and see whether any optimization is needed there. Good luck!

Maybe, transfer time is to short or long, depending on the device. Alternatively your Abs are not specific (for fre. see e.g. Baker, M. Blame it on the antibodies. Nature, 2015). Alternatively you (maybe accidentally) used the wrong type of membranes (nitrocellulose instead of PVDF), etc.

In general, I would propose to carefully check our working protocol and revise it. After that, you should repeat experiments and compare results to the case explained before.