blue native electrophoresis

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kriodos
kriodos's picture
blue native electrophoresis

I tried to run navive electroforesis for see if my purify protein is forming dymers. I tried a lameli-PAGE without DTT and SDS wich used to work but it doesn't work. My protein don't have enougth charge and don't penetrate the gel. I wan't to performe a blue native electrophoresis but I never tried this before. Someone can give me a easy protocol? I have my protein in PBS.
thanks!

qiang wang
qiang wang's picture
please check the link below

please check the link below for the blue-native gel protocol on-site
blue-native gel
 
also, I run before native gel adjusted from regular SDS-PAGE (no DTT or BME, no SDS), it worked pretty good, you will need to let the gel run for a longer time.
 
 

kriodos
kriodos's picture
thanks I didn't check the

thanks I didn't check the protocols. I will do it the next time
I tried the lammeli witoput DTT BME and SDS,  but it doesn't work at all. I thinking that maybe its not a dimerization but a polimerization. I addition the protein is quiete hydrophobic and withoout SDS the protein dont have the charge enougt at that pH for progress into the gel.
I,m going to try the blue navite
thanks again

qiang wang
qiang wang's picture
if you feel it could be

if you feel it could be dimerization, or even polymerization, make sure you run at low gel conc., I usaually run at 6-10%

Zipik
Zipik's picture
 Hi, i deal with purification

 Hi, i deal with purification of a unknown reductase. I need faoun enzymatic active band on electrophoresis after second purification step with help of stain  for its reducing activity (either UV determiantion NADP/NADPH or with tetrazolium salt)...do you have some experience if protein with bound coomassie are still enzymatic active? Or it is necessary use other method? Thank you for answer!

qiang wang
qiang wang's picture
So, what do you know about

So, what do you know about this unknow reductase?

Zipik
Zipik's picture
 That is the problem! We know

 That is the problem! We know almost nothing about its properties:-( We know only that is located in microsomes, its subtrate and its speterospecificity (and on this base we aretruing to purify it). So it is difficult to suggest native electrophoresis because we don't know anything about its charge and isoelectric point....so i found articles about blue native  electrophosresis and i want to use it as one possibility. I know that exist also colorless native electrophoresis, but it depends on its charge. We tried also classical PAGE electrophoresis without SDS, but there was no results.

qiang wang
qiang wang's picture
Zipik wrote:

Zipik wrote:

 That is the problem! We know almost nothing about its properties:-( We know only that is located in microsomes, its subtrate and its speterospecificity (and on this base we aretruing to purify it). So it is difficult to suggest native electrophoresis because we don't know anything about its charge and isoelectric point....so i found articles about blue native  electrophosresis and i want to use it as one possibility. I know that exist also colorless native electrophoresis, but it depends on its charge. We tried also classical PAGE electrophoresis without SDS, but there was no results.

 
Are you able to isolate microsmes?
<<We tried also classical PAGE electrophoresis without SDS, but there was no results>>
The protein could be membrane protein, then you will need mild detergent for the native PAGE.
You should be able to run BN-PAGE without the dye, then stain the gel with your substrates (you should be albe to find substrates that change color when react with the Reductase)
 
 

dcbayer04
dcbayer04's picture
To kriodos,

To kriodos,

I ran BN-PAGE for twice sofar, using Triton X-100 to dissolve my protein. The activity won't be lost even though the protein is bound to the coomassie dye. I use protocol from Ilka Wittig (2006), Nature Protocols.

Good luck
DC