Hello, I rely on your help. On February 27th, I prepared a Bradford reactive, left it for the night at room temperature, filtered it out on the next day and made a calibration graphic with egg albumen. An optical density of samples with different protein content constituted from 0.0237 to 0.0933. Ready Bradford reactive kept in the fridge then.

On 7th March I used this reactive for determination of protein content in Chlorella cells. OD constituted values, that were within ones of the calibration graphic.

Yesterday I did the same, but the values didn't differ and constituted 0.8430, something like that for all samples. I checked OD of plain Bradford reactive and it was just the same.

What might be wrong with the reactive? Do I need to prepare a new one? Or recalibrate the old? And what might such problems be connected with? I've never worked with Bradford reactive and I just don't know how to act.

Please, sorry for grammar mistakes, if there're they. I'm not a native English speaker.

55 minutes ago, Fanipal said:

Hello, I rely on your help. On February 27th, I prepared a Bradford reagent, left it for the night at room temperature, filtered it out on the next day and made a calibration graphic with egg albumen. An optical density of samples with different protein content constituted from 0.0237 to 0.0933. Ready Bradford reagent kept in the fridge then.

On 7th March I used this reagent for determination of protein content in Chlorella cells. OD constituted values, that were within ones of the calibration graphic.

Yesterday I did the same, but the values didn't differ and constituted 0.8430, something like that for all samples. I checked OD of plain Bradford reagent and it was just the same.

What might be wrong with the reagent? Do I need to prepare a new one? Or recalibrate the old? And what might such problems be connected with? I've never worked with Bradford reagent and I just don't know how to act.

Please, sorry for grammar mistakes, if there're they. I'm not a native English speaker.

 

57 minutes ago, Fanipal said:

Hello, I rely on your help. On February 27th, I prepared a Bradford reactive, left it for the night at room temperature, filtered it out on the next day and made a calibration graphic with egg albumen. An optical density of samples with different protein content constituted from 0.0237 to 0.0933. Ready Bradford reactive kept in the fridge then.

On 7th March I used this reactive for determination of protein content in Chlorella cells. OD constituted values, that were within ones of the calibration graphic.

Yesterday I did the same, but the values didn't differ and constituted 0.8430, something like that for all samples. I checked OD of plain Bradford reactive and it was just the same.

What might be wrong with the reactive? Do I need to prepare a new one? Or recalibrate the old? And what might such problems be connected with? I've never worked with Bradford reactive and I just don't know how to act.

Please, sorry for grammar mistakes, if there're they. I'm not a native English speaker.

 

Yes, I just wrote "reactive" instead of "reagent" 😖

You wrote, "An optical density of samples with different protein content constituted from 0.0237 to 0.0933."  Is this correct?  One problem that I see is that if your unknown has a larger absorbance than 0.0933, you will be doing an extrapolation, not an interpolation.

I generally filter the Bradford solution the same day as I use it.  When I want the highest accuracy, I prepare a standard graph on the same day as the unknown.