BISC220/S13: Mod 1 Lab 4

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Lab 1: Induction of β-galactosidase and Molecular Modeling
Lab 2: Affinity Chromatography and Specific Activity Determination
Lab 3: Enzyme Kinetics
The Michaelis-Menten Model
Media Recipes
Science Writing and Figure Design

Polyacrylamide Gel Electrophoresis

Another way to determine the effectiveness of our purification technique is to perform sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis on the CE and PF. SDS, an anionic detergent, is used to both denature the proteins and to give them an overall negative charge. The electrophoresis loading and running buffers also contain SDS. This negative charge allows the proteins to run toward the anode (positive electrode). The larger molecules are retarded by the acrylamide, which acts like a sieve, so they move more slowly through the gel than the smaller molecules. By running a sample of known molecular weights in a lane adjacent to the extracts, it is possible, by comparison, to determine the molecular weights of the bands of unknown proteins in each of the extracts. In addition to SDS, a denaturing and loading solution called Laemmli buffer contains:

  1. 10% glycerol, which makes the solution dense enough to fall through the running buffer into the wells of the gel, so the sample doesn't float away;
  2. 5% β−mercaptoethanol to break sulfide bonds and to keep disulfide bonds from reforming between the denatured proteins
  3. 2% SDS Sodium dodecyl sulfate,
  4. a trace amount of tracking dye, bromphenol blue, which will run ahead of the proteins, forming a visible front as the samples run during electrophoresis.
  5. in the solvent 0.0625 M Tris buffer pH 6.7

The gels are placed in the tank, which is half filled with cold running buffer. The top chamber is filled and checked for leaks, which can also cause distortion of the current. The current runs from the top chamber through the gel to the lower chamber, which is connected to the anode (red electrode). The negatively charged proteins will migrate toward the anode according to their molecular weights with the smaller proteins migrating farther down the gel than the larger proteins.


Microsoft Word File: Media:Gel Electrophoresis Protocol GelCode.doc

For SDS-PAGE, you will load 10 µl of each of your 4 samples (CE, PF, pre-IPTG cells, and post-IPTG cells) into lanes of a 4-15% gradient polyacrylamide gel. The crude extract and purified fraction should each contain 5 µg of protein.

  1. Make dilutions in Z buffer of your crude extract (CE) and your purified fraction (PF) to end up with 15 μg of protein in 15 μl of volume (1µg/µl concentration). You will have to look up the original protein concentration that you calculated for these samples. Remember that the sample of PF that you will use today has no added glycerol. **If you had less than 1 mg/ml of PF then you cannot make a dilution! See your instructor.
  2. Add an equal volume (15 μl) of Laemmli buffer, also called sample buffer. You will load 10 μl of this mixture onto the gel. The sample loaded contains 5 micrograms of protein. Your Instructor will demonstrate the loading procedure.
  3. In Lab I you saved some of the E. coli cells harvested before and after the β-galactosidase induction step with IPTG. To each of the 2 cell pellets add 75 μl of Laemmli sample buffer and resuspend the cells completely in the buffer.
  4. Boil all four samples for 5 minutes. Do not boil the molecular weight standard, but do boil the commercial β-galactosidase positive control along with your samples.
  5. Each electrophoresis chamber holds 2 gels, so 2 groups will run their gels together. Each group will load 6 lanes in one 4-15% gradient polyacrylamide gel according to the template below. Load 10µl of sample into each lane of your gel including a β-galactosidase positive control and the molecular weight standards. Be sure and take a copy of the key to the standards.
  6. Your instructor will connect the power source and set it to 200 volts. If no current reading (amps) appears, there is probably a leak in the upper chamber and extra running buffer has to be added to the upper chamber.
  7. After approximately 45 minutes, the tracking dye should have migrated to the bottom of the gel. TURN THE POWER OFF. Remove the gels from the reservoir. Your instructor will demonstrate how to remove the gel from between the plastic plates. Wear gloves when handling the gels because acrylamide is toxic.
  8. Place the gel in your plastic box and add enough deionized H2O to cover your gel and have it float freely. Incubate at room temperature with gentle rocking for approximately 5 minutes - repeat for a total of 3 times.
  9. Pour off the water and add enough GelCode Blue Stain Reagent to cover your gel and have it float freely. Return the gel to the shaker and stain for at least 30 minutes.
  10. To destain the gel add enough deionized H2O to cover your gel and have it float freely. Incubate at room temperature with gentle rocking for approximately 5 minutes - repeat for a total of 3 times.
  11. Photograph your gels using white light trans-illumination. Gel images will be posted to the conference as jpg files for incorporation into your lab reports.

Additional Calculations and Questions

Note: You do not have to turn in the answers to these questions as an assignment but you should incorporate the topics below into the discussion section of your lab report.

  1. Using measurements obtained from your gels plot the log10 of the molecular weights of the standard proteins (y-axis) versus mobility using the Instructions for Determining Molecular Weight.
  2. Using the standard curve generated in the previous step, calculate the molecular weight of the band(s) that you have identified as β-galactosidase. How well do your measurements agree with other published values for the molecular weight of β-galactosidase? Propose an explanation if your molecular weight differs significantly from the literature.
  3. Is the MW calculated above the same as that of the native β-galactosidase that you purified and assayed for enzymatic activity? Why/why not? How could you determine the answer to this question?
  4. Which of the bands on your gel exhibit the largest change in response to the addition of IPTG?
  5. Was β-galactosidase present before the addition of IPTG? How do you know?
  6. What other experiments could you do to prove that the band is β-galactosidase if your reader is unconvinced by the evidence of its migration at the same place as the standard?


The references cited here are on reserve for BISC 220 Cellular Physiology either electronically or in hard copy in the Science Library.

Background information on enzymes and lab techniques:
Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2002) Molecular Biology of the Cell 3rd ed. Garland Pub., New York.

Hawcroft DM (1997) Electrophoresis The Basics IRL Press, New York.

Kagedal L (1998) Immobilized Metal Ion Affinity Chromatography In Protein Purification 2nd ed. (Janson J-C and Ryden L eds) Wiley-Liss, New York.

Lehninger AL, Nelson DL, Cox MM (1993) Principles of Biochemistry 2nd ed. Worth Pub, New York.

Porath J, Carlsson J, Olsson I, Belfrage G (1975) Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258: 598-599.

Porath J, Olin B (1983) Immobilized metal ion affinity adsoprtion and immobilized metal ion affinity chromatography of biomaterials. Serum protein affinities for gel-immobilized iron and nickel ions. Biochemistry 22: 1621-1630.

Williams G (1998) HyperCell CD-ROM. Garland Pub., New York.

Specific References for β-galactosidase:
Jacobson RH, Zhang X-J, DuBose RF, Matthews BW (1994) Three-dimensional Structure of β-galactosidase from E. Coli. Nature 369; 761-766.

Juers DH, Hakda S, Matthews B, Huber RE (2003) Structural basis for the altered activity of Gly 794 variants of Escherichia coli β-galactosidase. Biochemistry. 42: 13505-13511.

Juers DH, Heightman TD, Vasella A, McCarter JD, Mackenzie L, Withers SG, Matthews BW (2001) A structural view of the action of Escherichia coli (lacZ) β-galactosidase. Biochemistry. 40: 14781-94.

Roth NJ, Rob B, Huber RE (1998) His–357 of β-galactosidase (Escherichia coli) Interacts with the C3 Hydroxyl in the Transition State and Helps To Mediate Catalysis Biochemistry 37: 10099-10107.

Ring, M, Huber RE (1990) Multiple replacements establish the importance of tyrosine 503 in β-galactosidase (Escherichia coli). Arch Biochem Biophys 283: 342-350