BSA Protein Standard Curve - Bradford Assay

BSA protein is reacted with Bradford Reagent to create a Standard Curve.
The chemical reaction is observed followed by the production of an Absorption Spectrum to determine the Amax. BSA Standards from 200 ug/mL down to the blank were used to generate the data. The Standard is plotted and used to determine the Concentration of 2 unknowns.
Protocol:
1.8 mL of Bradford Reagent was added to each Test Tube.
0.2 mL of sample with added to each.
They were incubated at RT (room temperature) for 5 min after gentle vortexing.
A Thermo Scientific Genesys 30 Visible Spectrophotometer was used to analyze the samples.

[@TheSashal1999]

Thanks for explanation! Much more clear than my professor who spent 20 minutes describing it and no one understood anything.

[@ProfessorDrewCollop]

Glad it helped!

[@taliaq3378]

If I have test sample contained 30% bio pharmaceutical and 70% host cell proteins, which standard protein would I use to estimate protein concentration? BSA or the biopharmaceutical reference standard?

[@ProfessorDrewCollop]

This assay just indicates how much protein is present, no matter what the protein might be. I used BSA as the protein standard in this experiment. Without knowing what the biopharmaceutical is, I cannot answer your question. Also, what is the purpose of the test sample? Are you planning to purify out the biopharmaceutical from the host cell proteins?

[@andyhinostroza3802]

Podrías hacer un video preparando reactivos de LOWRY

[@ProfessorDrewCollop]

Gracias por la consulta. No tenía planes de realizar un ensayo de Lowry, pero podría hacerlo en el verano, si lo desea. (Traductor de Google ya que solo hablo inglés)

[@prachibhatia8219]

how do you make the bradford reagent?. or what is bradford reagent made up of?

[@ProfessorDrewCollop]

Here is a recipe: www.usbio.net/protocols/bradfords-reagent

[@user-wd8tj6rb9w]

What are you using as your Blank?

[@ProfessorDrewCollop]

My solutions in this experiment contained 1.8 mL Bradford reagent + 0.2 mL of various concentrations of BSA. For my blank used in the standard curve, I used 1.8 mL of Bradford reagent + 0.2 mL of dH2O. This doubles as my 0 ug/mL solution in my standard curve. Alternatively, I could have used just dH2O as my blank for the absorption spectrum. This might change the Amax wavelength by a few nm from what I recorded. Hope this helps.