Hi Sandra,

I would use a high percentage gel (for example, 20%), and you could run it not for the end of the gel (you can even run its to the middle).

For detection it is better to use WB, and appropriate positive/negative controls, e.g. positive control for 6HIS (a proven protein with 6HIS), so you know that your antibodies are working; negative control – cell lysate free of the plasmid (a control for specificity), etc.

Also make sure that you follow the protocol for denaturation, sometimes you might not see monomers of the proteins due it dimerization, etc.

Good luck!

Hi Sandra, I hope you are well,

It is not easy to work with small proteins, especially because small proteins normally don’t have a stable tridimensional structure, and the folding of these small proteins is very difficult to carry out by the heterologous expression system, and as a consequence they typically are toxic for the host cell.

But assuming that you are obtaining overexpressed soluble protein, in order to see in Western Blotting and in coomassie staining polyacrylamide gels, there are some tricks that you have to take into account:

1.- Increase the crosslinking level of PAGE-SDS, normally we use a ratio of 36:1 of acrylamide: bis-acrylamide, use a ratio of 29:2 instead. This new stock of 30 % of acrylamide with an increase of the bis-acrylamide proportion will give you better resolution for smaller proteins.

2.- For Western Blot, if you are using an active electrotransfering equipment, as a semi-dry system, use the same current and voltage, but change the time of electro-transference at one third, for example, if you usually do the transference for 1 hour, just do it for 20 min, and check the transference with ponceau red. Smaller running times are better for smaller proteins because the membranes we use are of 0,45 μM pore size and the smaller proteins tend to go out the membrane after normal electrotransfering times used with other proteins.

I hope this could help you, greetings,

Ángel Lobo