Determination of the Molecular Weight of an Unknown Protein Using Size Exclusion Chromatography

By Andreea Stoichita and Chris Scanlon I. Introduction Size exclusion chromatography is commonly used in clinical and experimental labs around the world to separate proteins based on their molecular weight. (Carrier) Today, we will use it to determine the molecular weight of an unknown protein based on the time taken to move through the gel column, in our case a GE Superdex 75 10/300 GL column, as compared to the time taken to move through the column from a standard of 5 known proteins. We have mixed a standard of 5 proteins in solution that you will be using containing Aprotinin, Ribonuclease A, Carbonic Anhydrase, Ovalbumin, and Conalbumin. These proteins will move through the gel in the column interacting with the gel. The proteins with the higher molecular weights will travel through the fastest as they will interact less with the gel, as was discussed in class. (Chromatography) Once they pass through the column, they will pass through a spectrophotometer that is working at 280 nm, so you will get a readout on the computer when each protein is eluted from the column. There will be 5 peaks for the 5 proteins and it should look something like in Figure 1.
Protein UV Absorbance
160.00 140.00 120.00 100.00 80.00 60.00 40.00 20.00 0.00 -20.00 0.00

U A V bsorbance (m U A )

Series1

5.00

10.00

15.00

20.00

25.00

30.00

35.00

Volume Eluted (ml)

Figure 1. Elution profile of Standards on a Superdex 75 column using 50mM phosphate buffer at pH 7.0 containing 150mM NaCl at 0.5 mL/min. As you can see, there are 5 separate peaks. The overlap in the first two does not matter as this often happens with larger proteins. Finally, you will run an unknown protein through the column and determine its molecular weight by graphing the log of the Molecular Weight from the 5 proteins used in the first part vs. the kav, where kav=VE-V0/Vc-V0 (McKay) Through the chromatography you will obtain the VE. The VC is the volume of the column, which we have calculated to be Vc=*r2*h=24 mL. We also calculated the V0, or void volume, to be 7.65 mL. This was calculated by running a sample of Blue Dextran at 1mg/mL, an extremely high molecular weight molecule, through the column and determining the volume it took to elute it. II. Materials Used 1 mL Syringe Superdex 75 10/300 GL Size Exclusion Column High Performance Liquid Chromatographer

GE Gel Filtration Calibration Kit-Low Molecular Weight Buffer: 50 mM NaPi pH 7.0+150 mM NaCl 0.0197 moles NaH2PO4 0.0302 moles Na2HPO4 2.318 g NaCl Approximately 1 mL of distilled water Standard Solutions: 10 mg/mL Aprotinin in Buffer 20 mg/mL Ribonuclease A in Buffer 15 mg/mL Carbonic Anhydrase in Water 20 mg/mL Ovalbumin in Buffer 20 mg/mL Covalbumin in Buffer 1 mL Stock Solution: 300 uL Aprotinin SS (Standard Solution)+150 uL Ribonuclease A SS+200 uL Carbonix Anhydrase SS+200 uL Ovalbumin SS+150uL Covalbumin SS Unknown Solutions: 1 mL of Aprotinin Solution, Ribonuclease A solution, Carbonic Anhydrase Solution, Ovalbumin Solution, Covalbumin Solution 100 L tube pH meter micropipettes micro tubes 1 L beaker metallic spatula magnetic plate, magnetic rod, magnetic stirrer distilled water bottle electronic scales Components in Gel filtration calibration kits for LMW (low molecular weight) Protein (weight per vial) Aprotinin (AP) (10mg) Ribonuclease A (RA) (50 mg) Carbonic Anhydrase (CA) (15 mg) Ovalbumin (OV) (50 mg) Conalbumin (CO) (50mg) Blue Dextran 2000 Molecular weight (Da) 6,500 13,700 29,000 43,000 75,000 Concentration Source (mg/mL) 10 20 15 20 20 1 Bovine lung Bovine pancreas Bovine erythrocytes Hen egg Chicken egg white

III. Storage and Handling The kit should be stored at 2C to 8C. All chemicals should be considered potentially hazardous and should be used in accordance with the principles of good laboratory practice. Wear protective clothing, safety glasses and gloves. In the case of contact with the skin or eyes, wash immediately with water.

IV. Instructions for the preparation of the experiment (INSTRUCTOR ONLY) 1) Make the buffer from a weak base and a weak acid a. Wash a 1 L beaker with distilled water and add about 800 mL of distilled water b.Add a magnetic rod in the beaker and put the beaker on a magnetic plate c. Weigh 2.72 g of the weak acid NaH2PO4 (or 2.68 g of KH2PO4) and pour it in the beaker d.Weigh 4.29 g of the weak base Na2HPO4 and pour it in the beaker e. Weigh 2.32 g of NaCl and add it to the solution f. Add distilled water gradually to a volume of 1 L g.Stir the solution very well to dissolve the solutes check the pH of the solution using a pH meter (make sure you wash the pH tip with distilled water before inserting in the solution, and also make sure you keep the tip in the solution) h.If the pH of the solution is not 7 add some more NaCL solution drop wise i. Filter the buffer 2) Column is in ethanol (EtOH); calculate the column volume using the formula Vc=*r2*h=24 mL 3) Equilibrate the column a) Wash the column first with 2 column volumes of distilled water (start with 0.2 mL/min and slowly increase to 0.5 mL/min) b) Equilibrate the column with 2 column volumes of buffer (50 mM NaPi pH 7 + 0.15 M NaCl)

4) Calculate the Void Volume A. Insert a sample of Blue Dextran of size 0.5% of the column volume (100 L). NOTE: always apply a sample of 0.1-2% of the CV B. Calculate the void volume (Vo) by measuring the elution volume of the Blue Dextran C. Calculate the elution volume (Ve) by measuring the volume of the eluent from the point of injection to the center of the elution peak. 5) Create your standard solutions A. Make your standards in buffer except Carbonic Anhydrase which will be made in H2O B. Weigh 10 mg of Aprotinin and add it to 1 mL of buffer C. Weigh 20 mg of Ribonuclease A and add it to 1 mL of buffer D. Weigh 15 mg of Carbonic Anhydrase and add it to 1 mL of water E. Weigh 20 mg of Ovalbumin and add it to 1 mL of buffer F. Weigh 20 mg of Covalbumin and add it to 1 mL of buffer NOTE: Make sure you rinse the spatula with distilled water before each product weighing, and that you label the micro tubes with the name of the product, concentration, your name and the date! 6) Create your 5 protein mixture: with a micropipette measure 300 L of AP, 150 L RA, 200 L CA, 200 L OV, and 150 L CO and add them together to a total volume of 1

mL. Inject 100 L of the sample, and let the chromatographer run at a rate of 0.5 mL/min. 7) Check the pressure constantly 8) After the chromatographer finishes running export the data in an excel file and plot the mAU vs. mL. Calculate the eluted volume for each protein. Calculate Kav using the formula kav= (VE-V0)/ (Vc-V0) 9) Make the unknown samples in the following manner: Go to the instructions for the instructor only. Students can skip this part. V. Procedures for the groups to run experiment (FOR STUDENTS) 1.You will have a vial of the stock solution. Yours will have about 150 L of the solution in it. Remove the syringe from the chromatographer. Used the bottle with the distilled water to clean off the syringe. Fill the syringe with the entire volume of stock solution from the vial. Pull back on the stopper of the syringe very slowly. Make sure that there are no air bubbles in the syringe. 2.Return to the syringe to the entry point in the chromatographer, slowly pushing in until you feel it catch in the hole and you cannot push it any farther. 3.Slowly push the stopper in until all of the liquid has been injected into the 100 L tube. Do not worry about the excess. It will be collected in the waste container. 4.Begin a new run through the chromatographer on the computer. It will ask you to answer a series of questions. Fill in your name and the date. The column name is BMEN Lab, and the Book and Page is just Chromatography experiment. Obtain the name of the buffer from the materials used section, and the Solution volume is 100 L. Press next. You will be asked another mandatory question about the column. Type Same in this field. Press Next and then Finish. The chromatographer will now run on its own for approximately 55 minutes. As the chromatographer runs, there will be a graph that displays 3 different lines. The x-axis will be in mL, as in how many mL have been run through the column (for this experiment the solution will be run through at 0.5 mL/min). The three lines will represent UV absorbance, conductivity, and pressure and are color-coded. The UV absorbance graph is the one that we are interested in. The peaks in this graph will represent the times during the experiment at which the 5 different proteins were eluted. 5.Once the chromatographer is finished running, (it will beep when its finished, the x-axis on the graph on the computer will be labeled mL. The chromatographer will run until about 30 mL and the graphs of the UV absorbance, conductivity, and pressure will no longer be moving) you should see a graph for the UV absorbance spectrum, along with conductivity and pressure, that looks something like the graph from the introduction.

6.You will need to export this graph to Excel. To do this, click the tab that says Evaluation on the Start bar on the bottom of the computer screen (it will be currently minimized). Select the File tab and double click your file. It should be the first one in the column on the left hand side of the screen. Go to the toolbar File and select Export then in the Curves box select UV. Then press select and then export. It will take you to a Save As screen. Save the file onto your USB drive, making to save it as an Excel file by clicking the drop down box under the file name and choosing (*.xls). 7.At this point, you will inject the solution of the unknown protein. Change the syringe and wash the needle off with the buffer solution that is on top of the chromatographer. Inject the solution of the unknown protein using the same procedure as you did with the standard sample. 8.Start a new run on the computer using the same steps that you did in Step 4 and again wait for the chromatographer to run its course. 9.While you are waiting, go to one of the computers in the other room and open the File that you saved on your USB drive in Excel. You should have two columns of data, one in mL and one in mAU. Plot the data using a line graph. This should give you the same graph that you saw in the computer program before you exported it. From this graph and the columns of data, you should be able to tell the value for the mL eluted when each protein passed through the spectrophotometer. The number of mL that were eluted at the absorbance peak of each of the 5 proteins is your VE for each protein. Using this data, and the data in the chart below, you should be able to identify the peaks for each protein and calculate the kav for each protein. 10. Once you have done this, you can graph log of the Molecular Weight of each protein on the x-axis and the kav for each protein on the y-axis. Fit this data with a line of best fit using Excel. 11. When the chromatographer has finished with the unknown solution, follow step 6 to export the data and save it to your USB drive. That is all the data that you need to obtain from the laboratory. 12. Later, use the volume, in mL, that it took for the unknown protein to experience the absorbance peak. This value is your VE and can be used to calculate the kav of the protein. You can easily calculate the molecular weight of the unknown protein using the equation of the line of best fit that you obtained by graphing the log of the molecular weight of the proteins in the standard sample vs. the kav of those proteins. From the molecular weight, you should be able to identify the identity of the unknown protein from the given standard molecular weights. VI. Examples of Calculations Used Volume of Cylinder: Volume= *r2*h r=5 mm Volume= *5mm2*300mm

h=300 mm

Volume=23.56cm3=24mL Making Solutions of the Unknown Proteins in Concentrations of 3 mg/mL: Aprotinin (10 mg/mL originally): V1C1=V2C2 (1mL)(3mg/mL)=V2(10mg/mL) 300 microliters=V2 Ribonuclease A (20 mg/mL originally): V1C1=V2C2 (1mL)(3mg/mL)=V2(20mg/mL) 150 microliters=V2 Carbonic Anhydrase (15 mg/mL originally): V1C1=V2C2 (1mL)(3mg/mL)=V2(15mg/mL) 200 microliters=V2 Ovalbumin (20 mg/mL originally, mixed at a conc. of 4mg/mL): V1C1=V2C2 (1mL)(4mg/mL)=V2(20mg/mL) 200 microliters=V2 Conalbumin (20 mg/mL originally): V1C1=V2C2 (1mL)(3mg/mL)=V2(20mg/mL) 150 microliters=V2 Safety Information Buffer Ingredients Molecule or Solution NaCl Distilled Water NaH2PO4 Na2HPO4 Entire Solution

Safety Precautions Should not be ingested in a lab setting. No safety precautions necessarily. Not harmful to touch, however, should not be ingested. pH 4.4 Not harmful to touch, however, should not be ingested. Water solution is slightly alkaline. pH 9.0 Not harmful to touch, however, should not be ingested. pH 7.0

Proteins Protein or Solution Aprotinin Ribonuclease A Carbonic Anhydrase 6

Safety Precautions Do not touch with open skin. Should ingested. Used to reduce bleeding surgery. Not harmful to touch, however, should ingested. Not harmful to touch, however, should

not be during not be not be

Ovalbumin Conalbumin Protein Solution

ingested. Cannot be immediately dissolved in buffer. Must be first be dissolved in H2O. Not harmful to touch, however, should not be ingested in the lab setting. Same origin and precautions as ovalbumin. Not harmful to touch, however, should not be ingested.

For all other safety information and emergency protocol, consult the Material Safety Data Sheet (MSDS) that was included with the kit, for which you have a copy. VII. References Carrier, Rebecca and Bordonaro, Julie. Introduction to Chromatography. Introduction to Biochemical Engineering. 1994. Rensselaer Polytechnical Institute 18 February 2009 <http://www.rpi.edu/dept/chem-eng/Biotech-Environ/CHROMO/chromintro.html> Chromatography: Introductory Theory. Biosciences. 2003. Sheffield Hallam University. 17 February 2009 < http://teaching.shu.ac.uk/hwb/chemistry/tutorials/chrom/chrom1.htm> McKay, Patrick. An Introduction to Chromatography. National Health Museum. 2002. Department of Recovery Sciences. 17 February 2009 < http://www.accessexcellence.org /LC/SS/chromatography_background.php> Results to be reported: 1) Elution profile of the Standard with identification of each peak 2) Table with elution volumes (or times), molecular weight, Log MW, and Kav for each standard. 3) Example of calculations 4) Build a Calibration Curve with the standards. Use manual given (extra information given with the protocol) as an example of the type of curve you need to build. 5) Show equation and R2 6) Elution profile of your unknown sample 7) Identification of elution volume (or time) for your unknown 8) Calculation of the Kav for your unknown 9) Calculation of the Molecular Weight of your unknown using equation from cal. Curve 10) Identify your unknown sample Questions for the students: 1) Calculate the Resolution (Rs, as shown in class), for the first and the second peak. Comment 2) Calculate Rs for the third and fourth peak. Comment 3) What is the NaCl used in the elution buffer (mobile phase)? 4) What is the fractionation range for the column used?, what does that mean? 5) Explain with drawing what is: void volume, column volume, and bed volume