PROTEIN ASSAYS: BIO RAD AND PAGE GELS

Alice Franceschetti and Jessie Brantwein
1993 Woodrow Wilson Biology Institute

INTRODUCTION:

Concepts and techniques developed through the activities:

Bio Rad Assay

• Spectrophotometer use
• Sample Dilutions
• Micropipette use
• Selective dye utilization
• Preparing buffer solutions
• Graphing spectrophometric results
• Transmittance vs Optical density or Absorbance
• Calculations in mMoles
• Frequency and wavelength
• Plotting graphs according to the Beer-Lambert Law

PAGE Gels

Loading microsamples

• Photographing gels
• Measuring mobility of sample bands
• Calculations of MW in kDaltons
• Safety precautions to the teacher

Biotechnology Connection:

Bio Rad Assay: The biotechnology connection with this assay is mainly to learn the instrumentation procedures for the spectrophotometer and the micropipette both of which are invaluable tools in modern research. The graphing of the spectrophometer readings or any quantitative results is an indispensible tool which students must learn early in the AP and general Biology course.

PAGE Gels: Electrophoretic procedures, either vertical acrylamide or horizontal agarose systems, are increasingly being utilized for a variety of diagnostic, research and investigatory procedures. Again, graphing results against a standard is a valuable lesson.

Background Information:

When conducting laboratory assays for organic compounds, most Biology classes utilize a very simple protein test called the Biuret method. When combined with the protein in question, the blue Biuret reagent becomes a pale violet. However, this test does not indicate protein concentration or molecular weights.

One method for determining concentration is the Bio Rad Protein Assay which is a dye-binding procedure responding to various concentrations of soluble protein containing tyrosine, tryptophan or arginine resudes. Dilutions of the protein sample are made with a buffer such as Tris, and the Tris is used as a blank. The completed reaction is read on a spectrophotometer such as a "Spec 20" at 595 nm wavelength. Samples are plotted against a standard such as bovine serum albumin or bovine gamma globulin. A standard curve is set up plotting optical density against protein in µg/µl. For further information on this assay, one may investigate the Lowry (Folin-Ciolcalteau) or Bradford methods.

Molecular weights of unknown proteins may also be determined by the use of SDS polyacrylamide gel electrophoresis. When samples are run on "PAGE" gels, varying mobilities of proteins are due to the sieving effect of the gel. The ratio of acrylamide to bis-acrylamide sets up the size of the pores in the gel. One may run native gels with the protein intact, or one may denature with SDS, heat and _-mercaptoethanol. When the mobility of the sample proteins is plotted against a standard ladder, one may read the molecular weight in kiloDaltons. A ratio may be set up according to the mm's of mobility against the standard.

Target Group:

Bio Rad Assay may be used in general and more advanced biology and chemistry classes.

PAGE gels are probably geared more towards the AP Biology and Chemistry class, but the graphing against standard measures could prove beneficial to general courses.

Student/Class Time Required:

Students: The Bio Rad reagent is rather inexpensive since it must be mixed on 5:l ratio. If students are placed in groups it is advisable to put no more than two or three to a group or there is a tendency for some of the students to observe only. This procedure should take no more than one laboratory period if all members of the group are participating: one for dilutions and one for reading the spectrophotometer .

The PAGE gels should be cast by the teacher. Therefore, the protein preparation (denaturing) and dilutions may be completed by each group. It will take approximately two periods to complete this laboratory if the gels are previously cast. One period will be needed to denature the protein and one for running of the gel. The teacher may elect to stain with the Coomassie Blue.

Materials:

Bio Rad Materials:

Consumables

• 1 chicken egg
• Bovine Serum Albumin
• Tris-Cl Buffer
• Bio Rad Dye Reagent Concentrate
• Micropipette Tips
• Disposable Culture Tubes (Cuvette)
• BSA Standard

Capital Equipment

• Spectrophotometer
• Micropipettes
• Vortex

PAGE Gel Materials:

Consummables

• Ammonium Persulfate
• TEMED
• SDS (Sodium dodecyl sulfate)
• Tris
• Acrylamide
• Bis-Acrylamide -mercaptoethanol
• Glycerol
• Glycine
• Bromphenol Blue
• Methanol
• Acetic Acid
• Coomassie Brilliant Blue
• 30 cc syringes/needles Cost: About $5.00 per gel

Capital Equipment

• Micropipettes
• Mini-Protean Gel Chamber
• Power Source

Safety Precautions:

All solutions may be diluted and disposed of down the sink.

Student: Student should wear goggles and gloves for both the Bio Rad and PAGE gel procedures. Students should not cast gels; only deposit the samples in the wells.

Teacher: For the Bio Rad Assay, wear goggles and gloves. For the PAGE gel preparation, presence of a carcinogen and mutagen in acrylamide requires gloves, mask and goggles.

Teacher's Preparation Guide:

Bio Rad: Time required:

20 minutes to dilute and filter the concentrate

Solutions: 30 mls of Dye One Egg (Dye) 120 mls of DW Mix and filter Refrigerate if stored (Shelf life: several months if refrigerated)

Tris Solution: (50 mM) 25 ml of lM Tris (FW=121.1) 475 ml DW

PAGE Gel Solution: (Allow several hours for solution preparation and at least one hour to cast the gels. The gel will take several hours to run and several hours to destain.)

Stock Solutions Lower (Resolving) Gel 10% Acrylamide 0.25 M Tris, pH 8.8 0.2% SDS 0.25 mg/ml APS 0.l% TEMED	For Two Gels (For 15 ml mixture) 5.5 ml DW 5.0 ml Acrylamide Stock 4.0 ml 0.75 M Tris Stock 0.3 ml l0% SDS Stock 0.4 ml l0 mg/ml APS Add 15 µl TEMED (last) to initiate polymerization
Upper (Stacking) Gel 4% Acrylamide 0.15 M Tris, pH 6.8 0.2% SDS 0.25 mg/ml APS 0.l% TEMED	(For 10 ml mixture) 2.5 ml DW 1.3 ml Acrylamide Stock 6.0 ml 0.25 M Stock 0.2 ml 10% SDS Stock 0.25 ml 10 mg/ml APS Add 10 µl TEMED (last) to initiate polymerization
Electrode Buffer: 0.l M glycine 0.025 M Tris, pH 8.3 0.l% SDS	For 500 ml: 3.75 gm glycine 1.50 gm Trizma base 5.00 ml 10% SDS Stock Bring up to 500 mls DW
Sample Buffer: 0.05 M Tris, pH 6.8 6.8 1.5% SDS Stock 3.0% Glycerol 1.0% _-mercaptoethanol "Touch of Bromophenol Blue"	For 4X Buffer: 8.0 ml .25 M Tris, pH 1.5 ml 10% SDS 0.3 ml glycerol 0.4 ml _-ME Bromphenol Blue
Acrylamide Stock: 30 g Acrylamide 0.8 g bis-Acrylamide DW up to 100 ml (Store in dark and refrigerate)	Preparation of Sample: 250 µl 4X sample buffer 750 µl protein sample Boil 5 minutes in microtube

Use Bio Rad Standard SDS Page Low Molecular Weight Standards for your "Ladder".

Teacher's Procedure for Page Gels:

1. Dilute the yolks and albumin 99:1, DW to Sample.
   
2. Into a 1.5 ml plastic microtube, mix 250 µl of 4X sample buffer plus 750 µl of diluted sample (yolk or albumin) and place tube in boiling water for 5 minutes.
   
3. We used the Bio Rad Mini-Protean II System(#165-2940).
   
4. Mix and inject the upper, stacking solution and insert comb for well formation. Wait for polymerization.
   
5. Remove combs which formed the wells and insert samples. We used this pattern:

   1	2	3	4	5	6	7	8	9	Well #
   10	5	10	35	30	35	10	5	20	µl
   STD	Yolk	Yolk	Yolk	STD	Alb	Alb	Alb	STD	Sample

   
   
6. Run at a constant current of 10 mA until the tracking dye has almost run off the gel.
   
7. Remove the gel carefully and place in fixing solution for about 2 hours.
   
8. Pour off fixing solution and pour Brilliant Coomassie Blue over the gel for 4-8 hours.
   
9. Pour out Coomassie Blue and add Destaining solution for several hours. This may be repeated several times.
   
10. Use a Polaroid picture to aid in measuring migration of unknowns against standards.
    

Teacher's Outline for Presentation of Activity:

1. Introduction to Protein Molecules
   1. Four conformational groups of proteins
      
   2. Functions of four groups of proteins
2. Lab procedures for identification of proteins
   1. The Biuret Test (preliminary identification test)
      
   2. The Bio Rad Protein Assay: concentration in mgs/ml
      
   3. PAGE Gels: number and molecular weight in kDaltons of protein fractions
      
   4. Using the spectrophotometer
      
   5. Graphing molecular weights and concentrations of the proteins investigated
      
   6. Preparing solutions for the Bio-Rad test
      
   7. Using the micropipettor
3. Graphing the results
   
4. Calculations:
   1. Calculating in mMoles
      
   2. Arriving at concentrations and molecular weights by comparing with a standard

Student Instructions:

Bio Rad Assay:

1. 
   
2. Dilute both the yolk and albumin sample 99:l, DW to Egg
3. Five or six sets of dilutions with egg or BSA should be made with Tris buffer with three replications each.
   
4. Make a blank with Tris buffer. Use the table below for your dilutions.
   
5. Add the Bio Rad dye (diluted and filtered) to the samples. Wait at least 5 minutes.
   
6. Read all the samples at 595 nm
   
7. Plot the average readings of the yolk and albumin against the BSA curve.
   

(See hard copy for "Dilution Aliquots" Chart)

PAGE Gels:

1. Dilute both the yolk and albumin sample 99:l, DW to Egg
   
2. Combine 8 ml of Tris, 1.5 ml SDS, .3 ml glycerol, .4 ml _ ME and a touch of bromphenol blue. (This is your 4X buffer)
   
3. Add 250 µl of 4X buffer, 750 µl of the protein sample and boil 5 minutes in microtube.
   
4. Your teacher will have cast the gels in the Mini Protean Chamber.
   
5. Ust the grid below to determine the substance and its volume and into which well to insert the sample.
   
6. Your teacher will demonstrate how to fix, stain and destain your acrylamide gel.
   
7. Calculate the distance each band has travelled against the ladder standards. Then determine the molecular weight in kDaltons of each band.

(See hard copy for Page Gel Simulation)

protein2: Spectrophotometric Readings

(See "Results" Graphs on Hard Copy)

References:

Ausubel, F. M., et. al. Current Protocols in Molecular Biology, Volumes 1 and 2, John Wiley and Sons, New York, 1989.

Ausubel, F. M., et. al. Short protocols in Molecular Biology, John Wiley and Sons, 1989.

Bio Rad Protein Assay

Bio Rad Life Sciences Group

2000 Alfred Nobel Drive

Hercules, Ca. 94547

Clark, John M. Jr., and Robert L. Switzer, et al. Experimental Biochemistry ,(2nd ed.) W. H. Freeman and Company, New York, 1977.

Slater, Robert J., et al. Experiments in Molecular Biology, Humana Press, Clifton, New Jersey, 1986.

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