Experiment 1.1 - Introduction to the Biochemistry Laboratory

(updated on January 5, 2001)

OBJECTIVE

To determine the protein concentration of a biological macromolecule and estimate its molecular weight by gel electrophoresis.

EQUIPMENT

  • UV-visible spectrophotometer and disposable cuvettes
  • Mini-Protean 3 vertical gel electrophoresis rigs
  • Power supplies
  • Hot plates

REAGENTS

  • Biological Macromolecules
    • Lactalbumin, bovine milk, MW 14,175 Da
    • Lysozyme, chicken egg white, MW 14,307 Da
    • Myoglobin, horse heart, MW 16,900 Da
    • Chymotrypsin, bovine pancreas, MW 21,600 Da
    • Trypsin, bovine pancreas, MW 23,281 Da
    • Aldolase, rabbit muscle, MW 39,188 Da per subunit
    • Alpha Amylase, Bacillus species, MW ~50,000 Da
  • Tris free base
  • HCl
  • Bovine Serum Albumin (BSA) Standard, 2 mg/mL
  • Bradford Reagent
  • Solutions for Electrophoresis
    • 30% Acrylamide/Bisacrylamide Solution (CAUTION: Neurotoxin)
    • 1.5M Tris-HCl buffer, pH 8.8
    • 0.5M Tris-HCl buffer, pH 6.8
    • 20% (w/v) Sodium Dodecyl Sulfate (SDS)
    • 10% (w/v) Ammonium Persulfate (APS)
    • TEMED
    • 4X Tris-Glycine Electrophoresis Buffer (dilute to 1X before use)
  • Solutions for Coomassie Staining and Destaining
    • Methanol
    • Acetic Acid
    • Coomassie Brilliant Blue R-250
  • Gel Drying Solution (prepared by the TA)\

Abbreviations:

  • mcL = microliters
  • dI = deionized

REAGENT PREPARATION

1.5M Tris-HCl buffer at pH 8.8

  • Calculate the amount in grams of Tris free base (MW = 121.14 g/mol) required for 50 mL of 1.5M Tris base solution. Verify the accuracy of your calculation with the TA. Dissolve the appropriate amount of Tris base in <50 mL of dI water.
  • Measure the pH of the Tris base solution. Adjust the pH to 8.8 using HCl. (CAUTION: HCl is a strong acid. Wear gloves when handling this reagent and do not inhale this reagent.)
  • Do not exceed a total solution volume of 50 mL!
  • Add additional dI water as necessary to adjust the final solution volume to 50 mL.
  • Mix the solution. Label a storage container with the reagent name, concentration, measured pH, your name and the date of preparation. This reagent may be kept stored at room temperature.

0.5M Tris-HCl buffer at pH 6.8

  • Calculate the amount in grams of Tris free base (MW = 121.14 g/mol) required for 25 mL of 0.5M Tris base solution. Verify the accuracy of your calculation with the TA. Dissolve the appropriate amount of Tris base in <25 mL of dI water.
  • Measure the pH of the Tris base solution. Adjust the pH to 6.8 using HCl. (CAUTION: HCl is a strong acid. Wear gloves when handling this reagent and do not inhale this reagent.).
  • Do not exceed a total solution volume of 25 mL!
  • Add additional dI water as necessary to adjust the final solution volume to 25 mL.
  • Mix the solution. Label a storage container with the reagent name, concentration, measured pH, your name and the date of preparation. This reagent may be kept stored at room temperature.

20% (w/v) Sodium Dodecyl Sulfate (SDS)

  • Dissolve 1g of SDS in dI water to a final volume of 5 mL.
  • This reagent may be stored at room temperature. Crystals may form after long-term storage. Resolubilize any crystallized SDS by heating prior to use.

10% (w/v) Ammonium Persulfate (Prepare fresh)

  • Weigh out 0.1g of ammonium persulfate directly into a 1.5 mL microcentrifuge tube in an analytical balance.
  • Add 1 mL of dI water using a P1000 pipettor.

Preparation of 4X Electrophoresis Buffer

  • Dissolve 12g of Tris free base, 57.6g of glycine, and 4g of SDS in dI water to a total volume of 1 L.
  • At the time of use, dilute this solution 4-fold (to 1X) by adding 250 mL of 4X electrophoresis buffer to 750 mL of dI water.

Preparation of Staining and Destaining Solutions

  • Combine 125 mL of methanol, 25 mL of glacial acetic acid, and 100 mL of dI water to make 250 mL of destaining solution.
  • Dissolve 0.1g of Coomassie Brilliant Blue R-250 in 50 mL of the destain solution to make 50 mL of Coomassie staining solution.

PROCEDURE

  • Record the assigned protein for study in your notebook.
  • Dilute each sample 3-fold by adding 100 mcL of the protein to 200 mcL of dI water.
  • Perform the Bradford Protein Concentration Assay (see protocol under "Analytical Techniques" on the syllabus page) on the protein sample and determine the concentration of the protein sample. Be sure to account for sample dilutions.
  • Perform SDS-PAGE gel electrophoresis on the protein sample. (See the protocol under "Analytical Techniques" on the syllabus page.)
  • Perform Coomassie staining on the gel after electrophoresis. (See the protocol under "Analytical Techniques" on the syllabus page.)
  • Place two gel drying film sheets in drying solution. Neatly sandwich two or more gels between the wetted sheets within the drying frame. Secure the clamps onto the frame and leave drying gel at room temperature to dry.
  • Once the gel is dry, remove the frame. Tape the gel into your laboratory notebook.