There is a great need for a rapid and accurate method for quantitatively determining the protein concentration in a sample so that one can follow the recovery of proteins during the purification process. Although there is as yet no ideal method, the Bradford (1976) method is currently in very common use. This experiment was derived from the Laboratory Guide for Protein Purification and Identification by Houzhu Zhu.
Operation method
Quantitative measurement of proteins
Materials and Instruments
Bradford Reagents Bovine serum albumin (BSA) standard solution Tris-buffered salt solution Move Materials and equipment For more product details, please visit Aladdin Scientific website.
Microtitre plate Spectrophotometer or enzyme coupler
Bovine serum albumin (BSA) standard solution (2 mg/ml aqueous solution)
Bradford Reagent (CoomassiePlus, PierceChemicalCo.)
Spectrophotometer (capable of measuring at 595 nm) or enzyme coupler (with 595-nm filter)
Microtitration plate ( ImmulonTM2, Dynatech Laboratories, Inc.)
reagents
Tris-buffered salt solution (TBS)
(For recipes, see "Preparation of Reagents" PP.184~189)
Operating Procedures
1) Prepare a series of dilutions of BSA standard solution in TBS with the following BSA levels: 2000, 1000, 500, 250, 125, 62.5 and 31.25ug/ml. For each unknown sample to be tested, a series of doubling dilutions of the sample solution is also prepared in TBS.
2) Take 5ul of BSA dilution and unknown sample dilution and add 1ml of Bradford reagent to each. After mixing, incubate at room temperature for 5 min, and measure the A595nm value within 1 hour. The A595nm value can be measured more conveniently if an enzyme coupler is available. For the microtitre plate method, add 7ul of each sample to 200ul of Bradford's reagent, mix with a spiking tip and ensure that there are no air bubbles in the microtiter wells. If bubbles are present, puncture them with a 22-gauge needle. After 5 min incubation at room temperature, the A595nm value is determined.
3) Two controls can be measured. -One is to measure the various buffers present in the sample to see if they themselves develop color. The second is to measure an intermediate concentration range of BSA standards in the presence of different buffers to see if these buffers interfere with the color development of BSA.
4) Make a standard curve to determine the protein concentration of the unknown sample.
Note: Since some reagents (e.g., SKL, sodium deoxycholate, and polyethyleneimine) dumped during the σ32 purification process interfere with this method, we have found a microtitration method that can be replicated for this purpose, i.e., the assay was performed with 0, 5, 10, 15, and 25ug/ml of BSA and samples of the preparation diluted 100, 200, and 400 times. In each well of the microtitre plate, 100ul of Bradfod's reagent was added first, and then 100ul of each diluted sample was added, and then the above steps were followed.