BSA Protein Standard Curve - Bradford Assay

[@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!

[@aymentyba6335]

Can you please share the absorbance values obtained?

[@ProfessorDrewCollop]

Unfortunately, I will not be posting the Absorbance values. Students use this video to record down the % Transmittance and then they are calculated the Absorbances. If I post the Absorbance values, the students will not need to go through this part of the protocol. To calculated the Absorbance (A) from the % Transmittance (%T) use the following formula: A = 2 - log (%T)

[@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)

[@khushisaini9529]

What is the concentration of stock solution of BSA

[@ProfessorDrewCollop]

For this experiment you need to use 98% purified BSA. We buy the powder, so you can make up whatever concentration you would like. I believe for this experiment I used a 200 ug/mL stock solution of BSA.

[@Srirak-k3b]

what nanometer u have used for this protocol

[@ProfessorDrewCollop]

I don’t understand your question. Are you asking me what wavelength was used?

[@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?

[@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

[@данькаковырялка]

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.