[Jinal613]

This is a rather simple question and I feel really stupid for asking, but I'm just not getting it.

BSA standard is prepared in 0.15 M NaOH at a concentration of 1.0 mg/mL.
So there are a set of tubes and Tube 1, for example, has 10μL of the 1.0 mg/mL BSA, 40μL of the 0.15 M NaOH, and all the tubes have 2.5 mL of Bradford Reagent.
I need to calculate the Protein in μg/tube so I can plot that versus Absorbance on a standard curve.

So how do I calculate the protein concentration in μg/tube? I'm thinking μg/tube is basically μg/mL. But how do I calculate it?


Thanks.

[Jinal613]

Sorry correction. It's NaCl not NaOH.

Ok I tried doing it and this is what I got. I'm not sure if it's correct or not. If it isn't then some guidance would be great.

I converted 10μL BSA into 0.01 mL BSA.
The total solution (BSA + NaCl) is 50 μL = 0.05 mL. I didn't include the volume of the Bradford reagent as I thought the volume would be too small anyway..? Please correct me if I am wrong.

So this is how I did the calculation: 0.01 mL BSA in 0.05 mL solution

(0.01 mL x 1 mg/mL)/0.05 mL = 0.2 mg/mL
0.2 mg/mL x 1000 μg/mg = 200 μg/mL

But they wanted the concentration to be in μg/tube so I figured since 200 μg is present in just 1 mL and there is 0.05 mL per tube (total solution), I would have to multiply that amount by the total solution to get how much protein is there in the entire tube.

200 μg/mL x 0.05 mL/tube = 10 μg/tube  --> Answer!


Am I right in any of this, lol? Please let me know!

[Borek]

It is a little bit chaotic to me, but if

Tube 1, for example, has 10μL of the 1.0 mg/mL BSA

it contains 10x10^-3 mL x 1.0 mg/mL BSA. Other volumes would be needed for calculation of concentration.

[Jinal613]

Yes that's what I'm asking for. I need to calculate the concentration of the protein in each tube in order to plot it versus absorbance.

Tube 1 contains:
10μL of 1.0 mg/mL BSA
40 μL of 0.15 M NaCl
2.5 mL Bradford Reagent

Total solution in each tube would be 2.55 mL. But how do I calculate the concentration of protein in each tube?

[Borek]

You know how much, you know the volume, concentration is amount/volume.