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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

#1 Mariam

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Posted 02 September 2013 - 09:14 AM

Hi,

Since I have purified my protein of the interest using denaturing method, I had to get rid of urea before mice injection. I tried to dialyze my protein against reducing concentrations of urea in PBS. The dialysis has been done successfully as no aggregation happened. But when checking the protein concentration using Bradford assay, not only color change was not happened by adding the protein sample to Bradford reagent but also the concentration reported by the spectrophotometer was zero! However, after running the dialyzed protein on the SDS-PAGE the band of protein was visible! There were no detergent or other Bradford interferer in the protein elution buffer or in PBS. Could anyone help me with this problem?sad.png 

Thanks 



#2 labtastic

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Posted 04 September 2013 - 12:36 PM

Since you see the right protein on the gel but not in the bradford assay, either (i) the bradford assay was done incorrectly (did you do it side by side with a positive control, like BSA?), (ii) the protein is insensitive to bradford (some proteins don't work well with bradford...have you done it before on this protein and know that it should give a signal?), or (iii) you're not adding enough volume of your protein solution to the assay (use at least 1ug in a ml of reagent).

 

Alternatively, try the BCA assay. Works great.

 

Even better- since your protein is pure, why not just check absorbance at 280 nm?  Use the calculated extinction coefficient for your protein sequence and you should get a good idea of how much protein you have. 



#3 Mariam

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Posted 07 September 2013 - 09:06 AM

Since you see the right protein on the gel but not in the bradford assay, either (i) the bradford assay was done incorrectly (did you do it side by side with a positive control, like BSA?), (ii) the protein is insensitive to bradford (some proteins don't work well with bradford...have you done it before on this protein and know that it should give a signal?), or (iii) you're not adding enough volume of your protein solution to the assay (use at least 1ug in a ml of reagent).

 

Alternatively, try the BCA assay. Works great.

 

Even better- since your protein is pure, why not just check absorbance at 280 nm?  Use the calculated extinction coefficient for your protein sequence and you should get a good idea of how much protein you have. 

1) I performed the assay using standards (BSA) and the blank. 2)I have done the bradford assay for this protein before dialysis and it was ok. 3) I added 20 microliter  of the protein sample regarding the protocol. But after dialysis, I could not check the absorbance using bradford assay 



#4 labtastic

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Posted 08 September 2013 - 10:28 AM

Why can't you read absorbance at 280nm to determine protein concentration?



#5 Mariam

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Posted 09 September 2013 - 09:00 AM

Why can't you read absorbance at 280nm to determine protein concentration?

I did it.but the result was zero again! I think sth happens to my protein after dialysis that interferes with my protein but I can't find it!

Yesterday,I repeated the dialysis for some other sample of the same protein, this time although the result of the bradford was not zero,it was very low. but the band of the protein was sharp again on SDS-PAGE! 



#6 labtastic

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Posted 25 September 2013 - 05:12 AM

What is the amino acid content of your protein?



#7 Mariam

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Posted 13 October 2013 - 09:49 AM

Thanks for your reply.Sorry for my delay in responding. Here is the sequence of my protein: 

 

MADLAKIVEDLSALTVLEAAELSKLLEEKWGVSAAAPVAVAAAGGAAPAAAAEEKTEFDVVLADGGANKINVIKEVRALTGLGLKEAKDLVEGAPKAVKEGASKDEAEKIKAQLEAAGAKVELKETKVEWFGTVRARLGYTATERLMVYGTGGLAYGKVKSAFNLGDDASALHTWSDKTKAGWTLGAGAEYAINNNWTLKSEYLYTDLGKRNLVDVDNSFLESKVNFHTVRVGLNYKF



#8 mdfenko

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Posted 15 October 2013 - 04:57 AM

try reading at 215nm. this will read peptide bonds instead of ring structures (280nm).

 

are you sure the bands you see in the gel are your protein and not an artifact?


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#9 Mariam

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Posted 16 October 2013 - 07:42 AM

try reading at 215nm. this will read peptide bonds instead of ring structures (280nm).

 

are you sure the bands you see in the gel are your protein and not an artifact?

Thanks for your reply. Yes,I'm sure.It is single sharp band in the expected size! While reading at 215nm, should i add any reagent to my protein sample or it is like reading at 280nm by nonodrop?



#10 mdfenko

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Posted 17 October 2013 - 03:40 AM

you read it the same way as you read at 280nm. blank against whatever buffer the protein is in then read the sample.


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#11 Missle

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Posted 17 October 2013 - 04:09 AM

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)?



#12 Mariam

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Posted 17 October 2013 - 06:41 AM

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.

 

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? 



#13 Missle

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Posted 18 October 2013 - 03:55 AM

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?



#14 prabhubiograd

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Posted 18 October 2013 - 06:35 AM

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



#15 Mariam

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Posted 18 October 2013 - 06:59 AM

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?







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