Protein Quantification

1D, 2D, HPLC, SDS PAGE, etc. Talk about it here.
ssgrrens
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Protein Quantification

Postby ssgrrens » Sun Aug 04, 2013 11:10 am

Hello
I just performed bradford assay. My BSA stock is 1mg/ml. I did standard curve using 10, 20, 30, 40 and 50 ug (1mg/ml BSA stock). These are the values:
ug 0.D
10 = 0.21
20= 0.42
30= 0.69
40= 0.89
50 =1.05

Y = 0.0215x+0.007 and R2 = 0.9926

My sample O.D readings are below:
Sample 1= 0.28, sample 2 = 0.14, sample 3= 0.37


I extracted my protein and dissolved in 500 microlitres of IEF buffer: After I did my bradford assay, I got the following values:

ug/10 ul = 12.69767, 6.186047, 16.88372

ug/ul = 1.26, 0.61, 1.68

Does it mean my sample has 1.26 mg/ml? I have doubt because I dissolved in 500 microlitres of IEF buffer to dissolve my pellet (from this I took 10ul) and to store at -20C

I also need same amount (600ug) I can add upto 300-350 ul for for my IPG strips. But I got different values, Is there any formulae to calculate

Could you please answer to the above query

Thank you

tsbatth
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Postby tsbatth » Mon Aug 05, 2013 8:24 am

That looks right to me. What I would've done. Then again, wouldn't put too much thought into it, it will never be super accurate so just do enough replicates to get a consistent reading, also I wouldn't compare one set of sample concentrations from one day to another if you're trying to be really quantitative, better to do them side by side but you might just end up over thinking it IMO.

In regards to your question in the other thread about MS based proteomics, like any other tool (ie pcr, microarrays), is dependent on your research or project. If you think this platform will give answers and key insight compared to any other tool depends on what you're working on. I suggest some courses to have a better understanding of the technology and reading journals (Journal of Proteome Research, Molecular Cellular Proteomics, Proteomics, Science, Science signaling, Nature, Cell etc...), articles and reviews to gain a better idea of how the technology is utilized these days.

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Doug
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Postby Doug » Mon Aug 05, 2013 9:15 am

Actually I think this is a fine place to ask such a question. But I am not sure I understand the question. Did you dilute your BSA standards in the same concentration of IEF buffer as your samples? What is in your IEF buffer? There are a lot of components of buffers that also can be detected at the wavelengths measured for Bradford assay.
"If we knew what we were doing it wouldn't be research." -AE

tsbatth
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Postby tsbatth » Mon Aug 05, 2013 9:18 am

Fair enough, edited first part to what I wrote in PM.

ssgrrens
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Postby ssgrrens » Mon Aug 05, 2013 10:03 am

Yes. It's 8M urea, 4% Chaps, 40mM Tris-Hcl pH 8.5, 100mM DTT and 0.2% ampholyte.

Thank you. So does it mean my sample 1= 1.26 mg/ml? or 1.26 mg/500ul. The reason why I am asking this question because, I am confused a bit. My BSA stock is 1mg/ml. I did standard curve using 10, 20, 30, 40 and 50 ug (1mg/ml BSA stock).

My sample pellet is dissolved in 500ul and I took 10ul and did bradford assay.

If I increase the buffer to my pellet to 1ml, I am thinking that concentration will decrease. As such the O.D value will also decrease for 10ul (because it is more diluted) for my sample 1. But I am not sure if it is correct. Please correct if I am doing wrong.

Thank you

Biomarker
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Protein quantification

Postby Biomarker » Mon Aug 05, 2013 10:47 pm

Hello,

Yes you are right, your sample has 1.26 ug/ul conc. But if I remembered correctly you have used bradford from sigma (check the compatibility with your IEF buffer http://www.sigmaaldrich.com/content/dam/sigma-aldrich/life-science/proteomics-and-protein/migrationpep1/compatibility-chart.Par.0001.Image.-1.-1.1.gif. Comparing with the compatibility, you have used high conc. of urea, CHAPS, DTT). So I am not sure about your quantification results. And as Doug pointed out, did you dissolve your std in same IEF buffer?

ssgrrens
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Protein Quantification

Postby ssgrrens » Mon Aug 05, 2013 11:55 pm

Biomarker wrote:Hello,

Yes you are right, your sample has 1.26 ug/ul conc. But if I remembered correctly you have used bradford from sigma (check the compatibility with your IEF buffer http://www.sigmaaldrich.com/content/dam/sigma-aldrich/life-science/proteomics-and-protein/migrationpep1/compatibility-chart.Par.0001.Image.-1.-1.1.gif. Comparing with the compatibility, you have used high conc. of urea, CHAPS, DTT). So I am not sure about your quantification results. And as Doug pointed out, did you dissolve your std in same IEF buffer?


Yes, I dissolved in same buffer. However, my sample pellet is dissolved in 500ul and I took 10ul and did bradford assay and my stock of BSA is 1mg/ml. So my sample 1 has 1.26 mg/ml? not 1.26mg/500ul

If I increase the buffer to my pellet to 1ml, I am thinking that concentration will decrease. As such the O.D value will also decrease for 10ul (because it is more diluted) for my sample 1. But I am not sure if it is correct. Please correct if I am doing wrong.

Thank you.

Biomarker
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Protein quantification

Postby Biomarker » Tue Aug 06, 2013 12:13 am

Hello,

Yes according the volume you took i.e. 10 ul, your calculation is right that is 1.26 ug/ul. Do one thing to nullify the effect of IEF buffer. Start the expt with taking the 950 ul buffer and 50 ul miliq since you are making final conc to 50 ul while measuring std and sample. Take the OD of buffer, do the experiment in replicates for std as well as samples. And then subtract the OD of buffer from your std as well as sample.

I am not sure this is the correct way or not. Others please correct me if i am wrong.

Orelse switch to other IEF buffer.

Good luck

ssgrrens
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Postby ssgrrens » Tue Aug 06, 2013 12:35 am

Biomarker wrote:Hello,

Yes according the volume you took i.e. 10 ul, your calculation is right that is 1.26 ug/ul. Do one thing to nullify the effect of IEF buffer. Start the expt with taking the 950 ul buffer and 50 ul miliq since you are making final conc to 50 ul while measuring std and sample. Take the OD of buffer, do the experiment in replicates for std as well as samples. And then subtract the OD of buffer from your std as well as sample.

I am not sure this is the correct way or not. Others please correct me if i am wrong.

Orelse switch to other IEF buffer.

Good luck


Thank you. I will try this way and I will see if i get any difference

ssgrrens
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Postby ssgrrens » Tue Aug 06, 2013 4:33 am

Thank you. I didn't get any difference. I am just wondering, I am about to do a preliminary 2D experiment. I need 340ul (600ug). My sample has 1.26 ug/ul or 1.26 mg/ml. So 1260ug in 1000 ul. So if I need 600ug, Do I need to take 476ul from my sample? But I need 340ul. Sorry I am bothering you. We don't have any one in the lab to help.

ssgrrens
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Postby ssgrrens » Tue Aug 06, 2013 5:27 am

Sorry, I worked it out

Biomarker
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Protein quantification

Postby Biomarker » Tue Aug 06, 2013 5:56 am

Hello,

You can do lyophilization of your sample and then reconstitute it with 340 ul orelse you can speedvacc your sample to reduce it till ~340ul.


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