any idea about running small size of protein? - (Jan/23/2006 )
my protein is about 14kDa. I run it on normal 12%SDSPAGE. seems not good at all. anybody has idea that which kind of gel I can use for small molecular protein? or any commercial one I can use?
Thanks a lot
you should try 14 or 16% SDS-PAGE( tris-glycine)
I think that Tricine-SDS PAGE are used for separate small size proteins
Yes, very useful for less than 10 kDa.
In fact with tris tricine you get a better focus of your bands, and it helps to see small bands together with bigger one.
However if you are interested only by your 14 kDa proteins (and small proteins), you will manage perfectly just by increasing the percentage of you acrylamide. no need to change completely your protocol.
you can also check in invitrogen catalog. there is a figure somewhere where they explain which concentration of acylamide to use, for each size of proteins. You would see that with your 12%, 14 kDa is out of range.
I run a 12Kda protein on a 15% acylamide gel. Tris-glyceine works best for me. I did not get very good results with a Tricine based gel.
Run the gel for 30 mins at 200 Volts. Transfer in 20% methanol transfer buffer for 30 mins at 100V with an ice pack.
thank you all for useful anwsers. one stupid question, if 15% Tris glycine gel, do I just increase acylamide percentage and other components stay same as 10% gel?
yes you increase the quantity of acrylamide , other components stay same, unless water, that has to be decreased, depending on the volume increased of acrylamide, of course.
I mean, the final volume should be the same, then if you add more acrylamide, you should put less water to get the same final volume.