Introduction to the lab/safety features
A number of interesting and amusing suggestions (called Hints from Heloise) for performing experiments in the biochemistry lab (the hints are actually written for a genetics lab - but it's close enough) with care and precision have been made - check them out!!
Calibration of Pipetman
Each group will be given six Pipetmen: 2 - P20; 2 - P200; and 2 - P1000. These should be checked to be sure they measure the correct volumes. For each Pipetman, measure the mass of a delivered volume of water. Assuming the density of water is 1.00 g/mL, a direct check of the accuracy of the Pipetman is obtained. Repeat each determination three times (to check precision). Also check at three different volumes throughout the range of the Pipetman - such as 200 mL, 500 mL, and 750 mL for the P1000.
Experiment 1: Determination of Protein Concentration
Measure the absorbance, from around 220 nm to 350 nm, of the BPA standard solution on the photodiode array spectrophotometer. Dilute if necessary. Using the extinction coeficient at 280 nm, calculate the concentration of the solution. It should agree with the value given.
Also measure the absorption of the BPA solution of unknown concentration. Using the extinction coefficient, calculate its concentration.
Measure the absorbance, from around 220 nm to 350 nm, of the b-galactosidase solution on the photodiode array spectrophotometer. Dilute if necessary. Using the extinction coeficient at 280 nm, calculate the concentration of the solution. This is an unknown so no true concentration is given.
Remember that a spectrophotometric determination is non-destructive; in other words, the sample can be recovered and used for other experiments. So - be sure to recover the sample and don't dilute it more than necessary.
Prepare an appropriate volume of the Bradford working solution. Recall the stock is diluted - 1 part stock with 3 parts water.
Perform the Bradford assay using the BPA standard solution. Use the Hitachi spectrophotometers. Be sure to set the correct wavelength.
Perform the Bradford assay on the BPA and b-galactosidase solutions of unknown concentration. Be sure to perform the assay a sufficient number of times so that the precision is good and you are confident of the data.
Store any unused sample appropriately.