western blot protocols, troubleshooting, scientific resources and research atricles for the research community and science students


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Bradford Assay      
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a. Dilute Bradford dye concentrate with dH2O (see bottle for specifics, however 1ml dye + 4 ml dH2O is common)

b . Filter the dye solution (gravity filtration) – the diluted reagent may be used for approximately 2 weeks

c . A standard curve is needed to determine the concentration of the unknowns (i.e. samples)

d . In order to determine the protein concentration in the samples, you need to create a standard curve:

  

1. PubMed: comprehensive database - 15 million citations

2. NIH:the National Institutes of Health

3. UniProt: The world's most comprehensive catalogue of information on proteins

4. Western Blot Procedure Western blot protocol overview.

 

  
 



 

- Using 6 cuvettes (1ml capacity), add the following amounts of BSA: 0 (baseline), 2, 5, 8, 10, 15 micrograms

  
   

- Fill with Bradford dye (to a volume of 1 ml)

  
   

- Carefully vortex the cuvettes and allow them to incubate for approximately 5 minutes at room temperature.
Note: absorbance will increase with time, therefore be consistent with the amount of time that the samples are allowed to incubate before spectrophotometric measurement

  
   

- Using a spectrophotometer, measure the OD at 595 nm

 
e. Measure the OD of your samples; measure the OD of the lysis buffer as well to remove its contribution to total protein. 6. Plot the results for the BSA and make a standard curve

f. Using the curve, determine the protein concentration of your sample(s)
 
             
               
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