Zero absorbance by Bradford assay for Dialyzed protein - (Sep/02/2013 )
Your sequence has plenty of residues that should absorb quite well at A280. Have you tried dialyzing your protein into a different buffer? Do you have access to a different means of buffer exchange (like a zeba column or PD-10 column)?
mdfenko on Thu Oct 17 11:40:24 2013 said:
you read it the same way as you read at 280nm. blank against whatever buffer the protein is in then read the sample.
Thank you for your valuable tips.
Missle on Thu Oct 17 12:09:39 2013 said:
Your sequence has plenty of residues that should absorb quite well at A280. Have you tried dialyzing your protein into a different buffer? Do you have access to a different means of buffer exchange (like a zeba column or PD-10 column)?
No! we don't have such columns in our lab. But I have access to Amicon centrifugal filters. Do they work the same?
No, they don't work the same. The zeba or PD-10 columns are filled with a desalting resin that you equilibrate with your desired buffer and then add your sample and the protein flows thru into the new buffer. Zeba columns are made by Pierce and PD-10 columns are from GE - both their websites can provide background info. It's an extremely fast way to buffer exchange so you get your answers quickly but if your material is precipitated or precipitates during the process, then the resin can act like a filter and 'filter it out'.
Amicon centrifugal filters will work if you concentrate to a low volume and resuspend to full volume with your new buffer and repeat but the risk there is that you're protein is having periods of time of being highly concentrated and some proteins don't like that where others are fine.
Have you tried a buffer other than PBS?
Hi All,
I have also exactly the same problem. I have 3 (isoform)proteins of the same gene. I could express and purify them successfully. I checked on western blot and coomassie blue and they gave very good amount and purity. But when I concentrated using sartorius spin columns with 10,000Da cutoff or dialysed the protein, then I am unable to see the color change in bradford's assay. But the same protein gave some very high reading when I used nanodrop but again failed to give any reading with UV spectrophotometer at 280nm. I am confused how to proceed with.
I checked the protein again on western blot and coomassie blue and the protein is there. My proteins are very normal except the proteins 2 and 3 are heavily glycosylated. I don't think it will interfere with the readings on bradford or UV spec reading.
Please give me some suggestions.
Thanks
Prabhu
Missle on Fri Oct 18 11:55:49 2013 said:
No, they don't work the same. The zeba or PD-10 columns are filled with a desalting resin that you equilibrate with your desired buffer and then add your sample and the protein flows thru into the new buffer. Zeba columns are made by Pierce and PD-10 columns are from GE - both their websites can provide background info. It's an extremely fast way to buffer exchange so you get your answers quickly but if your material is precipitated or precipitates during the process, then the resin can act like a filter and 'filter it out'.
Amicon centrifugal filters will work if you concentrate to a low volume and resuspend to full volume with your new buffer and repeat but the risk there is that you're protein is having periods of time of being highly concentrated and some proteins don't like that where others are fine.
Have you tried a buffer other than PBS?
No,I did not. But some colleagues dialyze their proteins in a buffer which has the same reagents as the purification buffers:NaH2PO4, Tris-HCL or Nacl and urea.The major reason that I selected PBS was using the same buffers for all steps preparing recombinant protein for mice immunization.Since I am going to use PBS for recombinant protein diluting and as a positive control, I had to dialyze my protein in PBS. Can I change my dialyzing buffer?
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.....
Missle on Fri Oct 18 16:35:03 2013 said:
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?
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.
Missle on Wed Oct 23 12:19:40 2013 said:
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