Dear All,
I have purified 3 variants of the monoclonal antibody from CHO cell line expressing them, using 30 KD amicon ultra-4 centrifugal filter devices following by protein G purification, however when I run it on an SDS-PAGE gel, I get 2 bands of 70 KD and 55 KD instead of 50 and 25, to my absolute surprise!
I know that an antibody is 150 KD and when we boil it in the presence of DTT and run it on an SDS-PAGE gel we should get 2 bands, one for the heavy chain at 50 KD and the other one for the light chain at 25 KD (An antibody consists of 2 heavy chains and 2 light chains).
Would you please help me solve the problem?
Any comment or suggestion will be highly appreciated.
Best,
Niloufar
I would also propose to yue a low-concentrated SDS gele (about 10%) for 150 kD proteins.
SDS-PAGE is not a first principle method for molecular mass determination, some 20% error are quite normal. In addition, your gel is suboptimal: not well destained, with signs of band spreading (high salt?) and what is likely a keratin band across it. It also is poor practice to remove the stacking gel, you may need it to diagnose aggregation problems. Make sure that the sample volume in all wells is identical, using 1 x sample buffer.
Have you checked the protein in Native page? if you get the actual size then you can assume that the antibody is intact. and in denaturing page the 25kDa band is missing. so you can perform some binding assay with the supernatant and check whether the antibody produced is functionally active and the target is being identified. if this fails then the antibody is not assembling properly.
hi
As clearly seen from the resulting gel picture. There are a lot of errors had occured. As Dr. Engelbert clearly mentioned the unstain process was very poor. Have you loaded equimolar amounts in each well? Second i would like to add that the poor separation was not only due to buffer concentration but also regarding the protocol you have followed. Coulx you share with us how much % was the gel. And how much voltage have you used initially on start and final amount of time was the gel run. Another question have you performed any spectrophotometry ?
Regards
Kat.
Katerina Kouzari
Dear friend,
thanks for your consideration.
- No, the samples were not in same concentration even though I think the problem is not that !
- 12% gel
- 100 voltage
- No
Best
Beera Shankar Anand
Dear Beera,
thanks for your answer.
No I've not checked the native PAGE, I'm going to do that though.
what do you think about 10% gel instead of 12% I used?
Regards
Engelbert Buxbaum , i think 12% of gel is too much for a 150kda and this goes inversely with the volts which is 100v being used. I take it as a guess you didnt run the stacking gel initially at low . so i would suggest on initially run the stacking gel for 50-60v for approximately 25-30 mins. And after that perhaps increase at 120v. This for the improper separation. And regarding what Dr Engelbert Buxbaum said the destaining was improper also. It happens. check the links below. My question is how long did it took for your SDS PAGE?
Anything i have missed perhaps some else can add onto that
Best Regards
Kat.
https://www.cytographica.com/lab/protocols/gel_destain.html
You have to check your protein in Native PAGE also. As your gel picture, Run protein on 8 or 10% SDS PAGE for appropriate antibody separation, but initially in stacking gel you have run gel on low voltage (70-80v). Staining in not good as display in picture, so stain more for right size of protein. run gel 10-15 min more.
Niloufar Mohammadkhani Hi, your gel seems so condense and would suggest 10% gel or less. You also should be careful regarding the pH of staking and resolving gel. I would suggest prepare a pool of your sample and first the test of your new modified method then can run your samples. You might need also to increase the time of running. Moreover you can run beta actin or 12G10 as control to check your protocol.
Good luck!
Best,
Salman
I would also propose to yue a low-concentrated SDS gele (about 10%) for 150 kD proteins.
Yes, I would recommend you to use a lower gel concentration. Additionall as Beera says, why don't you attempt a Native PAGE. IF not, you may perfor MS of your protein sample.
Hello
Yes, I also advise you to use a lower gel concentration.
Good Luck
Hi, Niloufar Mohammadkhani
Maybe everything is already solved by now, but I couldn't see your pics. Have you took it off from the discussion? If so, is there any chancee to still look at it?
Lastly, please, whenever you solve your problem, please, send an answer showing your resolution to this problem in order to let others learn from your problem too. Ok?
Any help, feel free to contact us all ... :)
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