I have reduced the IgG4 to half antibody using TCEP. I see some (20%) free heavy and light chain (SDS-page), but they are still bound to each other (noncovalently). I see only one peak during purification. I was wondering how I can separate these from my half antibody or at least disrupt the noncovalent interaction between loose HC-LC.
Thanks.
Hi Dear Raheleh
Please send you sample buffer formula for me.
In SDS-PAGE, the protein must be fully denatured and non of the noncovalent bounds should not be existed.
1. Sample buffer formula?
2. Time of sample boiling?
3. The approximately light chain Mw?
4. The approximately heavy chain Mw ?
What mode of "purification" are you employing?
Have you tried Gel Filtration or Ion Exchange as an alternative to affinity (e.g. protein A, G). Sometimes non-covalent interactions can be modulated by running chromatographic approaches in different salt (high or low) concentrations. NaCL is a good salt to start.
You have good antibodies, the interaction between the chains talks about their possible stability. But their separation is in this difficult. To suppress interaction we use 4 - 6 M urea, you can be separated by gel filtration on Superdex or Sephacryl S100 HR Another way ion exchange chromatography on QAE media, or SP.
If you know the amino acid sequence you can be calculated pI of chains and to find the optimal pH and buffers for separation.
good luck
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