A few questions on SDS, coomassie staining & western - (Aug/10/2008 )
Hi
i have a few questions:
(1) Why do we make the mA a constant instead of voltage (i.e DNA electrophoresis) when we run
a SDS-PAGE gel?
(2) What is the mA that you guys use to run SDS-PAGE and western?
If you are transfering 4 westerns, would
you increase the voltage?
I use bout 500 mA (constant), about 50Vplus for them but the transfer looks bit faint?
Would it help if i increase the mA? I also reuse my transfer buffer.
(3) What's the purpose of acetic acid in the coomassie stain solution?
Also what is the coomassie ethanol solution recipe that you use?
Anyone knows what is the difference between sigma coomassie G and coomassie R?
Thanks in advance!
Heidi, I think I can answer at least your first two questions.
1) Different machines may require different voltages to obtain the same amps. Voltage (roughly) is the difference in electrical potential between two points and amps is the amplitude (height) of the energy waves. When you get electrocuted, higher amps are more likely to harm you than a higher voltage.
2) Although I should look at amps, I usually just run my transfers around 50 V. I don't know how many mA this corresponds to, but if you are getting faint transfers do make sure that your transfer buffer is clean without too much junk floating around in it. Also you should clean the tank with DI water before use so that there aren't extra minerals/ions contaminating your buffer. You may increase the amps as long as you make sure that your buffer/machine does not overheat...this is a common problem. There are many things you can do to correct this but the easiest is to put your transfer apparatus in a tub of ice during transfer. I also put a stir bar in there and stir it to equalize the temp.
Hope this helped. 
Thanks snazpistachio,
your answers did help.
Thanks for your tips. I do see some sponge debris floating around in the solution.
Probably that affected my transfer.
i usually do place a pack of icebag into my tank during the transfer. Thanks again
i have a few questions:
(1) Why do we make the mA a constant instead of voltage (i.e DNA electrophoresis) when we run
a SDS-PAGE gel?
(2) What is the mA that you guys use to run SDS-PAGE and western?
If you are transfering 4 westerns, would
you increase the voltage?
I use bout 500 mA (constant), about 50Vplus for them but the transfer looks bit faint?
Would it help if i increase the mA? I also reuse my transfer buffer.
(3) What's the purpose of acetic acid in the coomassie stain solution?
Also what is the coomassie ethanol solution recipe that you use?
Anyone knows what is the difference between sigma coomassie G and coomassie R?
Thanks in advance!
I have answer for your 3rd question.
methanol will precipitate the proteins in the gel in acidic condition. so acetic acid facilitate the precipitation. CBB-G has more sensitivity than CBB-R, but CBB-R provides greater resolution , so CBB-R is used in the SDS-PAGE. Due to its greater sensitivity CBB-G is used in Bradford assay. My staining solution uses methanol and the receipe is : 0.125% CBB R-250, 50% MeOH, 10% acetic acid.
[quote name='party' date='Aug 12 2008, 04:00 PM' post='146820']
Thanks party!
methanol will precipitate the proteins in the gel in acidic condition. so acetic acid facilitate the precipitation. CBB-G has more sensitivity than CBB-R, but CBB-R provides greater resolution , so CBB-R is used in the SDS-PAGE. Due to its greater sensitivity CBB-G is used in Bradford assay. My staining solution uses methanol and the receipe is : 0.125% CBB R-250, 50% MeOH, 10% acetic acid.
close, but not exactly.
acetic acid fixes the protein in the gel (you could use tca instead but if you don't wash out all of the excess then it will precipitate the coomassie).
cbb-r is more sensitive than cbb-g. cbb-g is more specific in its binding.
methanol will precipitate the proteins in the gel in acidic condition. so acetic acid facilitate the precipitation. CBB-G has more sensitivity than CBB-R, but CBB-R provides greater resolution , so CBB-R is used in the SDS-PAGE. Due to its greater sensitivity CBB-G is used in Bradford assay. My staining solution uses methanol and the receipe is : 0.125% CBB R-250, 50% MeOH, 10% acetic acid.
close, but not exactly.
acetic acid fixes the protein in the gel (you could use tca instead but if you don't wash out all of the excess then it will precipitate the coomassie).
cbb-r is more sensitive than cbb-g. cbb-g is more specific in its binding.
i use cbb-r for bradford...it works fine.