Experiment 1: Protein determination via the Bradford method
Bradford. Analytical Biochemistry. 72: 248-254. (1976)
This method is based on the idea that an acid solution of Coomassie Brilliant Blue G can be changed from 465nm too 595nm via protein binding. The resulting complex can be measured at 578nm in a filter photometer, so that the protein content of a solution after preparation can be used to create a calibration curve. The method was first described by Marion Bradford in 1976 and this is why it is so named. The method uses only one reagent and is quick and sensitive; it is also insensitive to buffers, reduction agents, and complex BILDNER, but is degenerated by bases and detergents. Numbers variations have been developed in order to increase the sensitivity and/or decrease errors caused by interferents. We will be applying reagents made by and a procedure developed by the Roth company.
Coomassie Brilliant Blue (CBB) is a tri-anilo-methane wool dye discovered at the end of the 19th century and is named after the West African city Kumasi (Coomassie in English; it is now in Ghana). It binds to bases (arginine most of all) and aromatic at a rate of approx. 1mg CBB per 1mg prtein. CBB-G250 is greenish with an anionic absorption maximum of 575-585nm and is used in the dection of proteins in experiments 1, 6, 8, and 12. Without aryl-methyl groups, CBB receives the additive R250 (reddish, with a maximum of 555-570nm) and is rarely used.
The protein contents of an unknown ß-gal-ase solution and an unknown bovine serum albumin (BSA) solution will be assesed using calibration curve created with a known protein concentration of bovine serum albumin.
You should have the following solutions at your disposal:
After creating the calibration curve, measure the following BSA dilutions into a test tube, using a graduated cylinder.
NOTE: The following steps involves contact with reagents containing H3PO4; be sure your safety goggles are securely fastened!
Into 7 different disposable half-micro (?) cuvettes, place 1 mL of each newly-made solution along with 1mL of the known ß-gal-ase solution and 250µL of the Roti-Quant 5x solution. The cuvettes should be labelled with a felt-tipped marker.
The measurements of the unknown BSA solution serves as an exercise in pipetting small quantities with an automatic pipette. These volumes should be taken 5x and the variaces recorded. Pay close attention to the following points, as they may throw off your experimental results: quality of the pipette, immersion depth of the tip, presence of droplets on the outside of the tip, presence of air bubbles, precipitate clumping in solution or on the walls of the reaction vesicle. Immerse your pipette tip in the unknown BSA solution and take up 1 µL. Deposit it in the cuvette (already containing the water), close with parafilm, and swirl, before adding the required amount of the Roti-Quant reagent and swirling again in the same manner.
All of the above preparations should be left to stand for 5 minutes before being measured into a 578nm filter for the Eppendorf photometer. The cuvettes must be used in such a manner that the thickness of the optical path must be 5mm, because of the high optical absorption of the container itself. The extinction should take place slowly over the next half hour; if the protein coagulates, no more useful measurements are possible.
Use the resulting values to create a caibration curve and characterize the concetration of the remaining ß-gal-ase; this will be used in a 1500x dilution in kinetics experiments 2 and 3 and thus, this experiment should be completed within the course of a single day. Hand in all 5 measured values, along with the average, standard deviation, and a discussion of sources of error in your lab report.
Copyright Misty Rious Productions, April 2003.