SDS-PAGE

Bands isolated from SDS-PAGE are frequently used for the production of antibodies to a particular protein. These bands are advantageous in that the isolated proteins are generally very pure. However, the antibodies raised against a protein isolated from SDS-PAGE gels will bind to denatured protein but may not recognize the native protein.

Below are two protocols for staining gels that allows the excision of the desired band without fixing the protein. This may not be practical for small (<10 kDa) proteins that could easily diffuse out of the gel. Small proteins should be bound to nitrocellulose.

Coomassie Staining (sensitivity ~ 2 micrograms)*

Wash gel with three changes of deionized water.
Add 50 ml 0.05% Coomassie Blue in deionized water
Incubate 10 minutes with gentle shaking at room temperature
Wash with several changes of water with gentle shaking for 3 hours or until the bands of interest are readily visible*
Excise the band(s) of interest with a clean razor blade and place in a cryo-vial or other tightly closed container.

Copper Stain (sensitivity ~ 0.1 micrograms)*

Wash the gel with five changes of deionized water
Transfer the gel to 50 ml 0.3 M Copper Chloride
Shake gently for five minutes
View bands against a black background (gel will turn white; protein bands will remain clear) and excise the desired bands using a clean razor blade.
Place gel fragments containing the desired protein in a tightly closed container.

* Adapted from Harlow & Lane. Antibodies a Laboratory Manual (1988)