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Zero absorbance by Bradford assay for Dialyzed protein

bradford assay Urea Dialysis Protein

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18 replies to this topic

#16 Missle

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Posted 18 October 2013 - 08:35 AM

I'm not sure if I am understanding correctly.  You are going to be using this protein as an immunogen for mice and also as the antigen in ELISA - is that correct?  If that is true, there is no reason you have to use PBS.  PBS is very common and I understand why it would be your first choice.  I don't know about your IACUC but you can immunize with urea in the buffer, especially if your protein is concentrated, as it will be diluted before immunization and likely mixed with adjuvant.

I don't know if changing buffers will help or not but it is worth a shot.

 

My only other suggestions is the same as labtastic's earlier - try a BCA.  It is significantly different than Bradford in mechanism (it detects amino acids 3+ in length as well as some individual residues).  I'm stumped though because Bradford's detection is the same as a standard SDS-PAGE, coomassie G-250 so it works in one but not the other.....



#17 Mariam

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Posted 19 October 2013 - 12:11 PM

I'm not sure if I am understanding correctly.  You are going to be using this protein as an immunogen for mice and also as the antigen in ELISA - is that correct?  If that is true, there is no reason you have to use PBS.  PBS is very common and I understand why it would be your first choice.  I don't know about your IACUC but you can immunize with urea in the buffer, especially if your protein is concentrated, as it will be diluted before immunization and likely mixed with adjuvant.

I don't know if changing buffers will help or not but it is worth a shot.

 

My only other suggestions is the same as labtastic's earlier - try a BCA.  It is significantly different than Bradford in mechanism (it detects amino acids 3+ in length as well as some individual residues).  I'm stumped though because Bradford's detection is the same as a standard SDS-PAGE, coomassie G-250 so it works in one but not the other.....

Yes. I am going to use my protein as an immunogen and also as the antigen in ELISA. I will try BCA.  However,as you mentioned, I can use my protein without dialyzing for mice immunization? am I right?



#18 Missle

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Posted 23 October 2013 - 04:19 AM

Yes I think so but some animal rooms do have a problem or guidelines around it so I would check first if I were you.



#19 Mariam

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Posted 23 October 2013 - 06:10 AM

Yes I think so but some animal rooms do have a problem or guidelines around it so I would check first if I were you.

Thank you very much for your kind support. Your tips helped me a lot.

 

Good luck







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