Devise a serial 5-fold dilution scheme into buffer A to dilute 2.82 mg/ml neuroglobin below A413 = 0.2 so that you can accurately read its absorbance. Remember you will need 1 ml of material at each stage of the dilution to read the mNgb dilutions in the spectrophotometer.
Given: You have a solution of 1 ml of murine neuroglobin (mNgb) that is 2.82 mg/ml according to a Bradford assay. The molar extinction coefficient at 413 nm of mNgb (ε413) is 82.1 mM-1 cm-1 and its molecular weight is 17, 600 Da).
- hcbiochemLv 74 months agoFavorite Answer
Put 1.6 mL buffer into 4 tubes. Add 0.4 mL of the original solution to the first tube. Mix. Remove 0.4 mL of that solution and transfer to the next tube. Mix. Remove 0.4 mL of that and add it to the third tube. And then repeat to the 4th tube.
The concentration of the 1st tube will be 0.564 mg/mL. The second will be 0.113 mg/mL, the third 0.0226 mg/mL and the 4th will be 0.0045 mg/mL
In the previous question, the absorbance of the stock solution was calculated to be 13.1. So, you can just divide that by 5 each time to calculate the absorbances of the dilutions:
1st dilution, absorbance = 13.1 / 5 = 2.62
2nd dilution, 2.62 / 5 = 1.31
3rd dilution, 1.31 / 5 = 0.262
4th dilution, 0.262 / 5 = 0.131
So, your 4th dilution will have an absorbance below 0.2.