10 - Bradford Assay

In this video, we use Bradford assay for protein quantification.
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[@mojzarea]

Thank you so much, Really helpfull

[@KIEUMANHUYTMU]

Dear Mister I am quite confuse at 13:24 . Does it mean value = 0.488 must be in between 0.43 and 0. 52 of the y axis? Thank you

[@georgenikolaras5609]

Shouldnt the curve be starting from the beginning of the two axes (point 0,0)? Because if you have no absorbance you cant have concentration. And also, isnt the standard curve a version of Lambert-Beer's law? If so, we cant have equation of y=ax + b formation, we will have the formation y=ax. And the thing i cannot understand is why we do the standard curve. Cant we just use the bradford reagent and measure the complex absorbance and after that, using Lambert-Beer's law we find the protein's concentration? Please correct me if im wrong.

[@paulinamontano5688]

As far as I understand for the Lambert-Beer's equation (A=C*e*l) you would need to use the extinction coefficient which is different for each reagent and is determined at a specific wavelength (defined by the solution you are measuring). If you observe, here they are using a 96 well plate here and is being read by the luminometer, I guess this is the instrument they will use to determine the concentration of proteins for their samples, but it can be done with a UV-vis. For the A=C*e*l normally you use an UV-vis spectrophotometer with a 1 cm cuvette, which allows you to set l=1 cm in the LB equation. (Or if you use a different pathway length I guess you would need to take that into consideration, for instance, if you are going to measure the concentration with a nanodrop, which you would need to adjust the value for "l", but anyway, I would strongly advise you to always use a cuvette to have more reliable results.

[@rawantej5139]

can you please explain how to prepare x and how to calculate the initial concentration of x in your exemple because i didnot realy get the calculation from the dilution

[@imranali-fm9sx]

How to prepare sample for maximum adsorption of BSA? Can you explain in detail

[@aishakhalfan2760]

that tube labelled X contain what?

[@NasarKhan4U]

can we check westernblot prepared samples with bradford? prepared sample mean samples with lemille buffer

[@fatimahamel9610]

I want a written rapport for this technique (materils,method, objectif and conclusion)pleaaaaaaase

[@nitikaaversatileworld4928]

How much Bradford reagent it's not audible

[@aziranafisahamiruddin313]

250 uL

[@freshd2431]

Great video! If you ever plan on recording a new version, you might add the standard deviation and error calculations to the Excel part of the video.

[@talebinas9406]

thank u

[@mehmoodulhassan4842]

sir I want it in written form can you provide it

[@leahmariabredenkamp7906]

Why do you only use the linear range?

[@nicolasromero8855]

Cause u can plot the linear data and get an ecuation from that. Then, u use that ecuation to determine the concentration of your protein. If u use non-linear data, would be harder to use a good plot ecuation

[@mohi7527]

Really great. But may I ask what is X?

[@bhaskarsharma9002]

It's very knowledgeable video