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Protein quantification


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#1 chandch

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Posted 04 August 2013 - 08:42 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

    

 

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 =  sample 1) 12.69767, sample 2) 6.186047, sample 3) 16.88372

 

ug/ul = sample 1)1.26, sample 2) 0.61, sample 3) 1.68

 

Does it mean my 1st 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


Edited by chandch, 04 August 2013 - 11:45 AM.


#2 doxorubicin

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Posted 04 August 2013 - 11:21 AM

The buffer you are using can influence the bradford.  Make sure that your blank and standard curve have the same final concentration of all buffer components as your lysate.



#3 chandch

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Posted 04 August 2013 - 11:38 AM

thank you. Is it not correct?  could you please explain it in detail? 


Edited by chandch, 04 August 2013 - 11:46 AM.


#4 Missle

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Posted 05 August 2013 - 05:18 AM

What were your OD values of your samples?



#5 chandch

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Posted 05 August 2013 - 05:44 AM

Here it is:  

 

Sample 1= 0.28, sample 2 = 0.14, sample 3= 0.37

 

 

Thank you






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