Bradford assay. Please check if I am done in a correct way

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ssgrrens
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Bradford assay. Please check if I am done in a correct way

Postby ssgrrens » Sun Jul 28, 2013 10:54 am

Hi all
* * I extracted plant protein using phenol extraction method by methanol precipitation. Could you please check if I am doing bradford assay standard curve and also checking protein concentration from unknown sample.


1) BSA Stock 1mg/ml dissolved in deionised water 2) Bradford reagent from Sigma

I took 5µl of stock BSA and add 995µl of Bradford reagent and checked 0.D at 595 nm and also I took 10µl, 25µl, 30µl, 40µl, 50µl, 60µl, 70µl of stock BSA and add 990, 975, 970, 960, 950, 940, 930 µl of Bradford reagent and did O.D at 595 nm.

BSA (1mg/ml) Bradford reagent 0.D 595
5 µl * * * * * * * * *995 µl * * * * *0.106
10 µl * * * * * * * * *990 µl * * * * *0.236
25 µl * * * * * * * * *975 µl * * * * *0.544
30 µl * * * * * * * * *970 µl * * * * *0.690
40 µl * * * * * * * * *960 µl * * * * *0.791
50 µl * * * * * * * * *950 µl * * * * *0.861
60 µl * * * * * * * * *940 µl * * * * *0.882
70 µl * * * * * * * * *930 µl * * * * *0.911

The value I got from the graph was

y = 16.04x

R² = 0.7773


2) Now I have my protein sample (unknown concentration), I did in the following way.


Protein Sample 1 Bradford 0.D 595
10µl * * * * * * * * 990 µl 0.117
40 µl * * * * * * * * 960 µl 0.726
50 µl * * * * * * * *940 µl 0.813

Protein Sample 2 Bradford O.D 595
10 µl * * * * * * * * 990 µl 0.135
40 µl * * * * * * * * 960 µl 0.545
50 µl * * * * * * * * 940 µl 0.821

Protein Sample 3 Bradford 0.D 595
10 µl * * * * * * * *990 µl 0.05
40 µl * * * * * * * *960 µl 0.321
50 µl * * * * * * * *940 µl 0.671

Then I did calculation in this way: Y = 16.04 x
x= y/16.04 and I got 0.958 mg/ml for sample 1, for sample 2 I got 0.904 mg/ml and for sample 3 I got 0.516 mg/ml

Now I need up to 350 µl for gels, but I need same concentration, so I took the lowest one which is sample 3 (516 µg in 1000 µl) so in 350 µl I got 180.6 µg.

Then I calculated for sample 1 (958µg in 1000 µl) so I got 180 µl and for sample 2 (904 µg in 1000 µl), so I got 199.7 µl


Is this the right way I did to check the known concentration and required concentration? Please help


Thank you
chandch

Biomarker
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Postby Biomarker » Sun Jul 28, 2013 11:54 pm

Hey,

Did you prepare your standard (BSA) in the buffer in which you dissolved your samples? And do check the comparability chart of the reagent.

I have used bradford reagent from biorad and it worked pretty well for me. The things which i noticed.

You have taken diff vol bradford for each std. I will explain u...

for e.g. if i m going to do std bsa expt with 10, 20, 30, 40 and 50 ug (bsa stock - 1mg/ml)
10 ul bsa + 40 ul miliq + 950 ul bradford
20 ul bsa + 30 ul miliq + 950 ul bradford
30 ul bsa + 20 ul miliq + 950 ul bradford
40 ul bsa + 10 ul miliq + 950 ul bradford
50 ul bsa + 950 ul bradford

incubate as per your time and take reading at 595 nm.

Similarly do follow for your sample with constant vol of ur bradford. Prepare your standard bsa in the same buffer in which u dissolved ur samples.

The R² must be > 0.95

Good luck with the expt..

cheers..
biomarker

ssgrrens
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Bradford

Postby ssgrrens » Mon Jul 29, 2013 12:08 pm

Thank you. I have dissolved BSA in deionized water (1mg/ml). I have dissolved my sample in isoelectric focusing buffer (8 M Urea, 4% CHAPS, 40 mM TriseHCl pH 8.5, 100 mM DTT, 0.2% ampholyte with trace bromophenol blue).

Is there any reason why I should dissolve BSA in same buffer? Can't I use distilled water for dissolving BSA?

Similarly do follow for your sample with constant vol of ur bradford? Does it mean do I need to use like this:

10 ul sample1 + 40 ul miliq + 950 ul bradford
20 ul sample1 + 30 ul miliq + 950 ul bradford
30 ul sample 1 + 20 ul miliq + 950 ul bradford
40 ul sample 1 + 10 ul miliq + 950 ul bradford
50 ul sample 1 + 950 ul bradford

and the same is followed for second unknown sample?

Could you please do sample calculation (4 unknown samples) for knowing the quantity from my unknown sample? If I have 4 sample, I may get different quantities. How do I calculate and use same concentration for experiments? I need 600 microg (300 microl) for my experiment. Please show it in excel, your sample calculation is highly appreciated. As our lab people are not experts in 2d electrophoresis.

Thank you

Biomarker
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bradford

Postby Biomarker » Mon Jul 29, 2013 11:09 pm

Hey,

U need to dissolve your std in the same buffer in which you dissolved your samples in order to nullify the effect of solvent.
And for sample, you should take the constant volume of your sample.
for eg.
10 ul sample1 + 40 ul miliq + 950ul bradford
10 ul sample2 + 40 ul miliq + 950ul bradford
10 ul sample3 + 40 ul miliq + 950ul bradford
10 ul sample4 + 40 ul miliq + 950ul bradford

note that absorbance at 595 nm.

I recommend you to use the bradford from biorad

ssgrrens
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Bradford

Postby ssgrrens » Tue Jul 30, 2013 12:20 am

Thank you. Could you please show the sample calculation from standard curve. For my experiment I need same concentration 600 microg (300 microl). Please show a example calculation, then I can do the same for my real experiment.


Thank you

Biomarker
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Postby Biomarker » Tue Jul 30, 2013 12:59 am

Hello, find the attached excel sheet. It is not the real data.

Good luck with your experiment

:)
You do not have the required permissions to view the files attached to this post.

ssgrrens
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Postby ssgrrens » Tue Jul 30, 2013 1:53 am

Thank you so much.

tsbatth
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Postby tsbatth » Tue Jul 30, 2013 4:31 am

Well to be honest, bradford assay is probably the least accurate method for protein concentration determination IMO. At best you'll get a ballpark estimate of the concentrations. I think this aspect of proteomics is highly overlooked. If concentrations are very essential (ie for targeted studies), from my experience florescent based assays or those based on lowry chemistry (ie biorad DC assay) works best. Also if exact concentrations are really important, you're better off doing quant on peptides rather then proteins. But for regular large scale proteomics workflows it should be ok.

ssgrrens
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Postby ssgrrens » Thu Aug 01, 2013 3:03 am

Thank you.


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