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protein estimation
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Sudheer Sangeetham
Apr 8, 2013, 4:57:29 AM4/8/13
to Met...@magpie.bio.indiana.edu
Hello all
I would like to quantify the protein concentration. I checked the nanodrop
manual in that they have given that I can estimate the protein by measuring
directly at 280 nm by giving extinction coefficient value without doing
bradford or lowry methods. I would like to choose measuring the protein
concentration directly. Because I need to handle many samples all the time,
so it is difficult for me to do bradford or lowry methods all the time. So
How far is correct if I estimate the protein concentration directly? please
give me your suggestion
Cheers
--
Sudheer Babu.S
Research Fellow
Institute of Biochemistry
Biological Research Center
Szeged,Hungary.
I would like to quantify the protein concentration. I checked the nanodrop
manual in that they have given that I can estimate the protein by measuring
directly at 280 nm by giving extinction coefficient value without doing
bradford or lowry methods. I would like to choose measuring the protein
concentration directly. Because I need to handle many samples all the time,
so it is difficult for me to do bradford or lowry methods all the time. So
How far is correct if I estimate the protein concentration directly? please
give me your suggestion
Cheers
--
Sudheer Babu.S
Research Fellow
Institute of Biochemistry
Biological Research Center
Szeged,Hungary.
Nick Theodorakis
Apr 8, 2013, 6:42:24 PM4/8/13
to Met...@magpie.bio.indiana.edu
On Monday, April 8, 2013 4:57:29 AM UTC-4, Sudheer Sangeetham wrote:
> Hello all
>
>
>
> I would like to quantify the protein concentration. I checked the nanodrop
>
> manual in that they have given that I can estimate the protein by measuring
>
> directly at 280 nm by giving extinction coefficient value without doing
>
> bradford or lowry methods. I would like to choose measuring the protein
>
> concentration directly. Because I need to handle many samples all the time,
>
> so it is difficult for me to do bradford or lowry methods all the time. So
>
> How far is correct if I estimate the protein concentration directly? please
>
> give me your suggestion
>
>
Do you have a mixture of proteins, or a purified protein of known extinction coefficient? The Nanodrop can be run in several modes (or at least some models can). For unknown mixtures, the best you can get is only a rough estimate (typically by assuming that a A280 = 1 corresponds to 1 mg/ml). If you have a pure characterized protein, some models will let you input the extinction coefficient and calculate it exactly. If you don't know the extinction coefficient but the sequence is known, there are online tools to estimate one, e.g.:
> Hello all
>
>
>
> I would like to quantify the protein concentration. I checked the nanodrop
>
> manual in that they have given that I can estimate the protein by measuring
>
> directly at 280 nm by giving extinction coefficient value without doing
>
> bradford or lowry methods. I would like to choose measuring the protein
>
> concentration directly. Because I need to handle many samples all the time,
>
> so it is difficult for me to do bradford or lowry methods all the time. So
>
> How far is correct if I estimate the protein concentration directly? please
>
> give me your suggestion
>
>
http://web.expasy.org/protparam/
Nick
--
Nick Theodoreakis
Message has been deleted
Message has been deleted
kaj.st...@helsinki.fi.invalid
Apr 9, 2013, 1:16:01 AM4/9/13
to
DK <d...@no.email.thankstospam.net> wrote:
> 1. If your protein is quite pure, A280 + theoretical extinction coefficient
> given by Protparam will *usually* get you as close to the real concentration
> as almost anything else (quantitative amino acid analysis is still a golden
> standatd but it's almost a lost art that few can execute competently and
> reliably these days).
> 2. If your protein is not very pure - particularly if contaminated by nucleic
> acids or large amounts of small molecules that absorb UV strongly, then
> just about anything else is going to be much more accurate than A280.
To expand a little more:
If the machine can reliably meassure at 205 nm that is the absorbtion
maximum of the peptide bond. Takes care of the problem with a protein
mixture. Small molecules and the buffer used might still pose a problem.
--
Kaj
> In article <mailman.236.136545...@net.bio.net>, Sudheer Sangeetham <sudhee...@gmail.com> wrote:
> >Hello all
> >
> >I would like to quantify the protein concentration. I checked the nanodrop
> >manual in that they have given that I can estimate the protein by measuring
> >directly at 280 nm by giving extinction coefficient value without doing
> >bradford or lowry methods. I would like to choose measuring the protein
> >concentration directly. Because I need to handle many samples all the time,
> >so it is difficult for me to do bradford or lowry methods all the time. So
> >How far is correct if I estimate the protein concentration directly? please
> >give me your suggestion
> To expand on Nick's answer a bit:
> >Hello all
> >
> >I would like to quantify the protein concentration. I checked the nanodrop
> >manual in that they have given that I can estimate the protein by measuring
> >directly at 280 nm by giving extinction coefficient value without doing
> >bradford or lowry methods. I would like to choose measuring the protein
> >concentration directly. Because I need to handle many samples all the time,
> >so it is difficult for me to do bradford or lowry methods all the time. So
> >How far is correct if I estimate the protein concentration directly? please
> >give me your suggestion
> 1. If your protein is quite pure, A280 + theoretical extinction coefficient
> given by Protparam will *usually* get you as close to the real concentration
> as almost anything else (quantitative amino acid analysis is still a golden
> standatd but it's almost a lost art that few can execute competently and
> reliably these days).
> 2. If your protein is not very pure - particularly if contaminated by nucleic
> acids or large amounts of small molecules that absorb UV strongly, then
> just about anything else is going to be much more accurate than A280.
To expand a little more:
If the machine can reliably meassure at 205 nm that is the absorbtion
maximum of the peptide bond. Takes care of the problem with a protein
mixture. Small molecules and the buffer used might still pose a problem.
--
Kaj
Vladimir Gainullin
Apr 9, 2013, 11:06:46 AM4/9/13
to DK, met...@magpie.bio.indiana.edu
Is it not true that after a standard curve is created (using a more
accurate method) one can use the A280 to estimate protein concentration
given that the sample is more or less similar?
On Mon, Apr 8, 2013 at 8:34 PM, DK <d...@no.email.thankstospam.net> wrote:
> In article <mailman.236.136545...@net.bio.net>, Sudheer
> Sangeetham <sudhee...@gmail.com> wrote:
> _______________________________________________
> Methods mailing list
> Met...@net.bio.net
> http://www.bio.net/biomail/listinfo/methods
>
accurate method) one can use the A280 to estimate protein concentration
given that the sample is more or less similar?
On Mon, Apr 8, 2013 at 8:34 PM, DK <d...@no.email.thankstospam.net> wrote:
> In article <mailman.236.136545...@net.bio.net>, Sudheer
> Sangeetham <sudhee...@gmail.com> wrote:
> >Hello all
> >
> >I would like to quantify the protein concentration. I checked the nanodrop
> >manual in that they have given that I can estimate the protein by
> measuring
> >directly at 280 nm by giving extinction coefficient value without doing
> >bradford or lowry methods. I would like to choose measuring the protein
> >concentration directly. Because I need to handle many samples all the
> time,
> >so it is difficult for me to do bradford or lowry methods all the time. So
> >How far is correct if I estimate the protein concentration directly?
> please
> >give me your suggestion
>
> >
> >I would like to quantify the protein concentration. I checked the nanodrop
> >manual in that they have given that I can estimate the protein by
> measuring
> >directly at 280 nm by giving extinction coefficient value without doing
> >bradford or lowry methods. I would like to choose measuring the protein
> >concentration directly. Because I need to handle many samples all the
> time,
> >so it is difficult for me to do bradford or lowry methods all the time. So
> >How far is correct if I estimate the protein concentration directly?
> please
> >give me your suggestion
>
> To expand on Nick's answer a bit:
>
> 1. If your protein is quite pure, A280 + theoretical extinction coefficient
> given by Protparam will *usually* get you as close to the real
> concentration
> as almost anything else (quantitative amino acid analysis is still a golden
> standatd but it's almost a lost art that few can execute competently and
> reliably these days).
>
> 2. If your protein is not very pure - particularly if contaminated by
> nucleic
> acids or large amounts of small molecules that absorb UV strongly, then
> just about anything else is going to be much more accurate than A280.
>
> DK
>
> 1. If your protein is quite pure, A280 + theoretical extinction coefficient
> given by Protparam will *usually* get you as close to the real
> concentration
> as almost anything else (quantitative amino acid analysis is still a golden
> standatd but it's almost a lost art that few can execute competently and
> reliably these days).
>
> 2. If your protein is not very pure - particularly if contaminated by
> nucleic
> acids or large amounts of small molecules that absorb UV strongly, then
> just about anything else is going to be much more accurate than A280.
>
> _______________________________________________
> Methods mailing list
> Met...@net.bio.net
> http://www.bio.net/biomail/listinfo/methods
>
Message has been deleted
Dr Engelbert Buxbaum
Apr 11, 2013, 12:45:51 PM4/11/13
to
In article <kk2stb$d56$1...@dont-email.me>, d...@no.email.thankstospam.net
says...
> to have this unfirtunate habit of being different from what we think of them :-)
>
> DK
Just to be fair, however, one should add that this is true for most
methods in routine use. Lowry, Bradford, BCA...: they all have
extinction coefficients that depend on amino acid composition of the
protein(s) analysed. I did that with Hsc70 once: depending on method and
standard protein used (BSA or IgG), the concentration determined varied
by a factor of 20! There are lies, damn lies and protein concentrations.
says...
>
> In article <mailman.243.136552...@net.bio.net>, Vladimir Gainullin <gain...@gmail.com> wrote:
> >Is it not true that after a standard curve is created (using a more
> >accurate method) one can use the A280 to estimate protein concentration
> >given that the sample is more or less similar?
>
> Yes. But the devil is in the "more or less similar" part. The samples tend
> In article <mailman.243.136552...@net.bio.net>, Vladimir Gainullin <gain...@gmail.com> wrote:
> >Is it not true that after a standard curve is created (using a more
> >accurate method) one can use the A280 to estimate protein concentration
> >given that the sample is more or less similar?
>
> to have this unfirtunate habit of being different from what we think of them :-)
>
> DK
Just to be fair, however, one should add that this is true for most
methods in routine use. Lowry, Bradford, BCA...: they all have
extinction coefficients that depend on amino acid composition of the
protein(s) analysed. I did that with Hsc70 once: depending on method and
standard protein used (BSA or IgG), the concentration determined varied
by a factor of 20! There are lies, damn lies and protein concentrations.
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