BSA Protein Standard Curve - Biuret Assay

BSA protein is reacted with Biuret 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 10 mg/mL down to the blank were used to generate the data. The Standard is plotted and used to determine the Concentration of 2 unknowns.
A Thermo Scientific Genesys 30 Visible Spectrophotometer was used to analyze the samples.

[@shirishzambre5751]

This is really helpful..thanks a lotot

[@ProfessorDrewCollop]

Glad it was helpful!

[@aqeedatrasool7044]

Good afternoon professor, Thank you for the video. I just want to ask aren,t we suppose to Blank eachtime before we record %T for every standard and unknown using live display mode?

[@ProfessorDrewCollop]

You only need to blank if you change the wavelength. If you are using the same wavelength, you just need to blank it the one time. Hope this helps.

[@again5t.your53lf6]

Biurette may be good for larger concentrations, bradford is much more sensitive

[@ProfessorDrewCollop]

You are correct. That is why I have another video featuring the Bradford assay.
ua-cam.com/video/Ool9Vair-O0/v-deo.html
We usually start of with first semester students using the Biuret assay, then switch to the Bradford when they gain more experience in second semester.

[@hustler-music]

what if I'm trying to measure the efficacy of a protease at varying pH levels. I'm conducting a biuret test for each condition, but what wavelength should I pick to measure the solution? Online many things indicate 540nm, why is this? Is it because it is most contrasting to purple ?

[@ProfessorDrewCollop]

Hi Fabian. When using a Spectrophotometer you want to always set the wavelength to the one of maximal absorbance (Amax or λmax). This will provide you with the largest dynamic range between max and min absorbance. This will ensure the data you are collecting has the best resolution. To determine the Amax, create an Absorption Spectrum using a positive control. This value can usually be found online; however, I always determine it during an experiment to double check. The Genesys 30 Spectrophotometer can perform this automatically in just a few minutes. I hope this answers your question and sends you on the right path.

[@afuri2843]

hello sir, thanks for video.. i want to ask how to determine precentage protein of the sample? is there a specific formula? thank you sir 🙏

[@ProfessorDrewCollop]

The Standard Curve is used to determine the concentration of protein in an unknown sample. You compare the data obtained from the unknown sample, to the data obtained from the Standards. The Standards are sample that you make up, so you know the concentrations. It is extremely important to have strong lab skills when making up the Standards. If your Standards are incorrect, it will be impossible to correctly determine the concentration in the unknown sample. Hope this helps.

[@afuri2843]

Thank you so much sir for explained, that's so much help!!

[@affafkherief185]

hello sir, please tell me how can i prepare a BSA stock solution, then how to dilute it to 10%?

[@ProfessorDrewCollop]

When you ask me how to prepare a stock solution, you need to provide me with a concentration. You can directly prepare a 10 % (w/v) solution by adding in 10 g of BSA per 100 mL of solvent.

[@Abhinavkr147]

Why is bsa used for?

[@ProfessorDrewCollop]

Are you asking what BSA is used for in this particular experiment, or what is it used for in the lab generally?