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.
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.
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
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
This is a gorgeous learning resource thank you so much. I wish there were more demonstrations like this of lab protocols online.
How to prepare sample for maximum adsorption of BSA? Can you explain in detail
Thank you so much, Really helpfull
can we check westernblot prepared samples with bradford? prepared sample mean samples with lemille buffer
I want a written rapport for this technique (materils,method, objectif and conclusion)pleaaaaaaase
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.
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.
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
that tube labelled X contain what?
How much Bradford reagent it's not audible
250 uL
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.
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
sir I want it in written form can you provide it
thank u
It's very knowledgeable video
Really great. But may I ask what is X?
Why do you only use the linear range?
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