2.3: Day 3 Procedure - Protein Assay

Day #3: Protein Assay of a Sample of Catalase from Bovine Liver Culture

In this part of the experiment, the student will learn about pipetting with a pipetman and also calibration of the pipetman (see Appendix 1). In addition, each student will prepare a standard curve, practice a serial dilution, and operate the UV-VIS spectrometers.

Students will receive a sample solution of catalase enzyme from bovine liver culture. Students will use the Coomassie Plus Modified Bradford Protein Assay to determine the concentration of the catalase in the sample.

Each pair of students assembles a tray of microcentrifuge tubes: five tubes containing 50 micro liters each of unknown catalase solution, and seven empty tubes for mixing standard solutions the zero standard will serve as your blank. Students will be provided with a bottle of 40mM Tris HCl buffer, (pH 8.2).

Experimental

Calibration of the Pipetman

For proper operation of the pipetman please see (Appendix 1). Calibration of the pipetman is as simple as dialing up a volume on the instrument. Drawing up that amount of water from a beaker and pipetting it into a tared weighing vessel. Then, reading off the mass from the balance and knowing the density of water 1.00 g/mL, calculating the volume. Compare this to the volume dialed up on the instrument. This will indicate the accuracy of the instrument. From our experience with this experiment, most pipetman are accurately calibrated and in cases where they are not, they are generally off by no more than ±1µL.

Coomassie Protein Modified Bradford Method

The basic principle of the modified Bradford method works as follows: The Coomassie Plus dye in its acidic red-brown form absorbs at 465 nm (A₄₆₅). When the dye forms a complex with the protein, it is converted to an anionic blue form with the absorbance shifting to 595 nm (A₅₉₅). The absorbance is directly proportional to the concentration of protein present. Students first generate a standard curve using the protein Bovine Serum Albumin (BSA) by measuring the absorbance at 595 nm of a series of seven standards of known concentrations. Next, student will measure the A₅₉₅ of the student’s five samples and determine their concentration by interpolating the reading at 595 nm against the standard curve.

Instructors will provide the following solutions:

Buffer 40mM Tris HCl (pH 8.2): Dissolve 4.8456 grams Tris in 1 liter Milli-Q water acidified with HCl to pH 8.2.

Coomassie Plus Protein Assay Reagent: 950 mL Product #1856210 Thermo Scientific

Albumin Standard (BSA): 2 mg/mL, 50 mL bottle, Product #23210 Thermo Scientific

Catalase: Worthington Biochemical Corporation, Catalase 0.22µm Filtered Code: CTS 10 mL vial of catalase with exact concentration. Instructor should dilute with 10 mL 40 mM Tris HCl buffer to create the unknowns for student samples. Worthington Product Code #LS001896

1. Preparation of BSA standards¹⁵

Prepare a set of seven Bovine serum albumin (BSA) protein standard solutions by diluting the 2.0 mg/mL BSA stock solution as illustrated in Table 1. When you have finished making A & B, snap-lock the tubes mix well and centrifuge them for 30 s. Then make up Standards C & D from A & B as below, snap-lock all four tubes mix well and centrifuge them for 30 s. Now make up Standard E from D, snap-lock both tubes mix well and centrifuge for 30 s, finally make up F from E, and G. Snap-lock mix well then centrifuge for 30 s. Follow the procedure steps indicated below. The centrifuge machine must be balanced please do not operate it if not balanced or the motor will burn up. If you are not sure about how to balance the centrifuge please check with your TA.

Table 1: Preparation of BSA Standards

Volume/(µL) of the BSA to Add	Volume/(µL) of Diluent (Buffer) to Add	Final Volume/(µL)	Final BSA Concentration/(mg/mL)
300 of Stock	0	300	Stock - 2000
375 of stock	125	500	A - 1500
325 of Stock	325	650	B - 1000
175 of A	175	350	C - 750
325 of B	325	650	D - 500
325 of D	325	650	E - 250
325 of E	325	650	F - 125
0	325	325	G - 0

2. Preparation of the Coomassie Plus Protein Assay Reagent

Obtain a bottle of Coomassie Plus reagent from the refrigerator. Mix the Coomassie Plus reagent by gently inverting the bottle twice. Pour out the amount of reagent that you need for the lab. Each pair of students will need about 25 mL. Allow the poured Coomassie Plus reagent to come to room temperature 40 minutes prior to adding it the samples and standards.

3. Procedure

1. Pipette 50 µL each of seven BSA standards (A thru G) and 50 µL each of five unknown catalase samples into twelve labeled microcentrifuge tubes.
2. The BSA ‘G’ Standard above will also serve as your blank.
3. To each of the seven standards and five unknown samples, working quickly, add 1.5 mL of Coomassie Plus reagent. Snap lock the microcentrifuge tubes and gently mix by inverting them several times. Allow the tubes to sit for 5 minutes at room temperature for color to develop.
4. Transfer standards, unknown samples and blank to separate 1.5 mL plastic disposable UV cuvettes by gently mixing then, opening the microcentrifuge tubes and pouring each by hand into the 1.5 mL plastic disposable UV cuvettes. Immediately proceed to the UV-VIS spectrometer to measure the absorbance at 595 nm for each standard and sample vs. blank. It’s important that you run your samples within 10 minutes (no later than 15 minutes) of adding the first drop Coomassie dye. No more than 15 minutes should elapse between the time you add the Coomassie dye and the complete running of all the standards and samples. Generally, it takes about 2.5 minutes to add the dye to all 12 standards and samples. Snapping the tubes shut and mixing takes another 2 minutes. Opening and pouring the tubes into UV cuvettes takes 2 minutes. At 9 minutes, you should be in the UV room running the air blank on the instrument then your zero blank followed by loading the standards. When your stop watch reads 10 min press the start button running your standards this takes about 2 min, then take out the standards and load your samples you should be at about 13.5 min, press start and run the samples finish at 15 minutes. Follow the guidelines for starting the program and operating the UV-VIS Spectrometer (see Appendix II).
5. Chart a linear standard curve using the BSA standards in the range of 125 µgml-1 to 1000 µgml^-1 by exporting data to Microsoft Excel and do a linear regression curve fit by plotting the average blank corrected absorbance 595 nm reading for each BSA standard vs. its concentration in µg/mL. Do not force the computer generated linear regression through zero. A Bradford Assay has three linear regions when BSA is used as a standard. One of the linear ranges is from 0 to 125 µgml^-1 another is from 125 µgml^-1 to 1000 µgml^-1 or 125 µgml^-1 to 750 µgml^-1 [Test these to see which gives the steepest slope] a third is from around 1000 µgml^-1 to 2000 µgml^-1. The value of the sample absorbance reading determines which linear range is used to calculate the protein present in the sample. As absorbance increases the accuracy of the value decreases. It is advisable to assay using protein concentrations that fall on the linear line with the largest slope. Please verify this using either 125 µgml^-1 to 1000 µgml^-1 or 125 µgml^-1 to 750 µgml^-1 (Option Two: If electing to use all of the standards 0 µgml-1 to 1500 µgml^-1 and comfortable working with absorbance values greater than 1, chart a standard curve by exporting data to Microsoft Excel and do a curve-linear regression using a 3-parameter polynomial curve fit by plotting the average blank corrected absorbance 595 nm reading for each BSA standard vs. its concentration in g /ml)
6. To determine the unknown protein concentration, interpolate the absorbance values from each unknown sample against the standard curve.

Clean-Up:

Empty all of the leftover solutions into the appropriate Catalase waste container. The empty UV cuvettes must be disposed of in the plastic waste container. All the plastic pipette tips and microcentrifuge tubes should also be disposed of in the plastic waste container. If you are not sure, please ask your TA. DO NOT PLACE THE PLASTIC UV CUVETTES OR THE PLASTIC PIPETTE TIPS IN THE BROKEN GLASS WASTE BOX.

Analysis and Discussion

1. Determine the protein concentration of your unknown sample. Discuss reasons in general why performing a protein assay is important.
2. Do a detailed error analysis on your unknown concentrations including standard deviations, error of the mean, and 95% confidence levels for the results.
3. Include a line of best fit for the data in the laboratory appendix that clearly shows a correlation coefficient (R value).
4. Discuss any random and or systematic error that could be present and any other sources of error that are present.

Footnotes

¹⁵ Adapted from: Dolhun, J. J., 5.301 Chemistry Laboratory Techniques, January IAP, 2012. (Massachusetts Institute of Technology: MIT OpenCourseWare). https://ocw.mit.edu/courses/chemistry/5-301- chemistry-laboratory-techniques-january-iap-2012/ (accessed Mar 27, 2013). License: Creative Commons BY-NC-SA