PROTEIN STRUCK IN SDS PAGE

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dogramicrobiology
dogramicrobiology's picture
PROTEIN STRUCK IN SDS PAGE

Hello Sir/ Madam,
                                      i have  prepared the bacterial outer membrane proteins by ultra centrifugation method
  and stored in phosphate buffer saline(pH7.6) -20*c.I have started doing SDS PAGE of these samples according to Sambrook's protocol. 5%stacking gel (pH6.8), 12% resolving gel(pH8.8), tris glysin buffer (pH-8.3),  2x sample buffer (50mM tris, 2%SDS, 0.1% BPB, B-MERCAPTO , 10% glycerol).
 I am running gel at 100mV const. voltage. by doing all described above i have been facing folowing promlems.

1. the major problem is,max. fraction of loaded  protein  struck either in stacking gel or in resolving gel and I could not get proper band separation. important point to take  into cosideration is that SDS PAGE RULAR  gave very clear protein  band separation in same gel.

SMEARING AND SMILING ARTIFACTS ALSO TAKES PLACE

if any body have solution to my problem then please help me.
 

Chin Fen Teo
Chin Fen Teo's picture
 dogramicrobiology,

 dogramicrobiology,

Given the description of your problem and that your sample buffer obviously does not include glycerol, please double check and include glycerol in your sample buffer per the standard protocol. The sample can't migrate down the gel well in the absence of glycerol in the sample buffer.

Good luck. 

dogramicrobiology
dogramicrobiology's picture
thanks for your kind

thanks for your kind information about glycerol. but actually i forgot to mention thwe glycerol here. i was already adding 10%glycerol in my sample buffer.
 so kindly give some other solution to my problem.
 thanks

Rajiv Dua
Rajiv Dua's picture
Dear Dogramicrobiology,

Dear Dogramicrobiology,
        what i feel is u need to check the pH properly of  your running buffer as well of  gel buffer , the  stacking buffer pH (6.8) plays a important role , try 4% stacking with 14 or 16% resolving gel depends upon the mol.wt of protien of interest....

Hope this may help u...

Cheer'
RD 

Chin Fen Teo
Chin Fen Teo's picture
 dogramicrobiology,

 dogramicrobiology,

Based on your gel image, your samples did enter the gel, only that the separation was not one would expect.

Having seem that the problem is restricted to your samples (it look rather like buffer/salt effect), yet you got relatively decent separation with your protein marker, the next thing I suggest you to do is (1) dilute your sample may be 5 to 10 fold to see whether you can improve the resolution, (2) in the case where dilution is not an option due to low amount of sample to reach detection threshold, you may try to precipitate your samples (with methanol/chloroform or acetone or TCA, which ever is more convenient in your mind), then boil them with the sample buffer and load onto the gel.

Good luck.

dogramicrobiology
dogramicrobiology's picture
thank you all who have

thank you all who have replyed to my problem
 
I have followed your instructions, but by doing so only my marker and standard albumin protein could gave good resolution on SDS PAGE . but my outer membrane proteins of bacteria(pasteurella multocida )which is asarcosyl detergent insoluble prepration. And then stored in phosphate buffered saline (ph 7.4). at -20*c since last six months. when initially i have run sds page of these proteins i got caparitively good bands. but now after 6 months trying with same protocol i did not get good protein bands.protein become precipitated during storage. is there any problem with this precipitation. how could i get rid of this problem. please suggest me some good solutions.

 thank you very much

Chin Fen Teo
Chin Fen Teo's picture
 dogramicrobiology,

 dogramicrobiology,

It sounds like the cause of your problem is because of your samples, instead of any potential technical issues.

If you can see the precipitant in your sample tubes, try to heat it up and see whether it can get back to the solution before loading onto the gel. Otherwise, I think you have no choice but prepare a new batch of the sample and analyze it quickly...

Good luck.

dogramicrobiology
dogramicrobiology's picture
 hello to all

 hello to all
 i have prepared fresh batch of outer membrane protein of pasteurella and this time i have dessolved the protein pellet in the sterile deionized water  then protein was run on the 12% SDS PAGE as described above . but problem of stucking of protein in  gel remains as it is. i am attatching some photographs of gel . please give me suitable solution according to your experience. 

     hope fully i will get a good solution
 thank you all.