How to determine the protein concentration????
I have NADPH in my protein sample so the problem is with the spectophometer I can not determine the protein concentration.
I shall be very thankful to you for your kind reply.
with best regards
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4 replies to this topic
#2
madrius1
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Posted 24 March 2009 - 06:06 AM
Not sure I get your point..
You have no reading on your spectrophotometer? On a Bradford assay i suppose?
And/or you have a signal when blotting for NADPH?
You have no reading on your spectrophotometer? On a Bradford assay i suppose?
And/or you have a signal when blotting for NADPH?
#3
mdfenko
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Posted 25 March 2009 - 06:24 AM
are you sure that the nadph will interfere with reading the bradford and/or lowry and/or bca protein assays?
if so then you may want to try fluorescamine. it is a fluorescent protein assay that should not be affected by nadph.
why is there nadph in your protein solution?
if so then you may want to try fluorescamine. it is a fluorescent protein assay that should not be affected by nadph.
why is there nadph in your protein solution?
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#4
samita
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Posted 26 March 2009 - 01:29 AM
I am expresing a protein in E.coli and purified it through affinity blue column and elute it with high concentration of NADPH. I dialyse it with buffer for mass spectrometry but they told me NADPH will not be removed from the protein through dialysis step so the protein concentration is not accurate through spectrophotometer.......
mdfenko, on Mar 25 2009, 06:24 AM, said:
are you sure that the nadph will interfere with reading the bradford and/or lowry and/or bca protein assays?
if so then you may want to try fluorescamine. it is a fluorescent protein assay that should not be affected by nadph.
why is there nadph in your protein solution?
if so then you may want to try fluorescamine. it is a fluorescent protein assay that should not be affected by nadph.
why is there nadph in your protein solution?
#5
T C
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Posted 26 March 2009 - 02:52 AM
Hey,
In that case, purify it further by FPLC and use the buffer that you want to do yr mass spec in .
Alternatively, do a PD-10 desalting and elute in the buffer of yr choice.
Best,
TC
In that case, purify it further by FPLC and use the buffer that you want to do yr mass spec in .
Alternatively, do a PD-10 desalting and elute in the buffer of yr choice.
Best,
TC
samita, on Mar 26 2009, 03:59 PM, said:
I am expresing a protein in E.coli and purified it through affinity blue column and elute it with high concentration of NADPH. I dialyse it with buffer for mass spectrometry but they told me NADPH will not be removed from the protein through dialysis step so the protein concentration is not accurate through spectrophotometer.......
mdfenko, on Mar 25 2009, 06:24 AM, said:
are you sure that the nadph will interfere with reading the bradford and/or lowry and/or bca protein assays?
if so then you may want to try fluorescamine. it is a fluorescent protein assay that should not be affected by nadph.
why is there nadph in your protein solution?
if so then you may want to try fluorescamine. it is a fluorescent protein assay that should not be affected by nadph.
why is there nadph in your protein solution?
