I use SDS page sample buffer recipes to liver rat to determine carboxylesetrase enzyme using SDS PAGE. The recipe are Tris HCl 0.125 M; SDS 4%; DTT 0.15 M; glycerol 20% and Bromphenol blue 0.01%.
I got this recipe from a paper, but it didn't explain how is the protocol?
Do I have to mix all of them at the same time?? OR Some mixed early, and then the others??
Would you give me some suggestion?
Thank you very much
This looks like a recipe for a SDS loading buffer, 4 or 5X concentrated. All the components are soluble in water, so just decide which final volume you want and start adding one component after the other to water. I would suggest you to add the bromophenol blue at the end as it is quite intense and tends to stick on the surfaces of your beaker/bottle.
As far as my experience goes, you can initially mix the Bromophenol blue & Tris.Cl (pH 6.8- since that is the pH of your stacking gel) , vortex them together in your microfuge tube, thereafter add the SDS solution & DTT at the respective concentrations & vortex them. Add the required volume of glycerol to get the final concentration of 20% v/v only at the end & vortex the contents thoroughly so that the lighter & denser phases mix properly.
Please refer to the following link for the Laemmli's method of electrophoresis
http://www.bio-protocol.org/e80
Alternatively to DTT, beta-mercaptoethanol could also be used since it serves the same purpose of breaking disulfide linkages for SDS-PAGE.
However if you are interested in the measurement of in-gel carboxyl esterase activity , please refer to the protocol for native PAGE as given in Analytical Biochemistry 315(2):208-22 · May 2003 among other sources. They have used a fluorometric procedure therein.
The recipe you give a 4 x sample buffer. Simply add 1 volume of that to 3 volumes of sample, mix and heat. Although 5' in boiling waterbath are standard, many proteins give better results with 15' at 60°C. Btw, do not replace the DTT with ß-mercaptoethanol. DTT (Cleland's reagent) was specifically designed to have the right redox potential for breaking S-S bridges in proteins. ßME is a lot weaker (and besides smells awful ;-( )
Your suggestions are very usefull, thank you very much Dear Domenico Sanfelice; Sebastian Reichau; Mrinalini Menon; Engelbert Buxbaum
One question
On that paper mention that in some cases if the protein is very hard to purify such as membrane, it is necessary to add some non ionic detergent such as Triton X-100.
What is the function of Triton X-100?
Should I add Triton X-100 on my recipes?
As per Dr.Bauxbaum's inputs as well as the information given on http://www.bio-rad.com/en-in/faq/268440261/technical-support-faq , I understand that where 5-10 mM DTT would be used, we shall need about 100 mM BME as the latter is highly volatile & might not bring about complete reduction of disulfide bridges.
Triton X -100 is more effective and powerful detergent than SDS for denaturing the proteins and providing an overall linear structure. However, going with Triton X-100 in the beginning is not advisable. First try to run your sample in a normal SDS gel and if still you face problem in obtaining clear bands, then you can again opt for Triton.
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