a. Microzone EP cell 1. Cell block w/ 2 buffer reservoirs (w/ electrode) 2. Baffle plates control convection currents 3. Bridge holds membrane in position 4. Cell cover has positions for multiple sample application 5. Cell lid prevent loss of H2O by evaporation b. Duostat supply power supply; regulates voltage/current 1. Meter Upper Left 2. Meter switch UR; allows meter to be used as a voltmeter (voltage) or ammeter (current) 3. Function switch LR; selects either constant voltage or current as the output 4. 2 push buttons: red & black represent 2 coded leads from the back of the Duostat to the cell; check individual cell current
When neither button is pushed Total current is
measured 5. Output adjust switch turns on the power; advance the needle to the desired output c. Sample applicator w/ red (retract) & white (pick up) buttons d. Electrophoresis membranes Advantages of Cellulose Acetate: 1. Shorter running time 2. Better resolution 3. Smaller sample size 4. 8-24 samples simultaneously 5. Can be cleared e. Blotters, forceps, squeegee, plastic envelopes, parafilm Reagents: a. Beckman B2 or Veronal buffer - pH 8.6 - = 0.075 b. Fixative-dye solution (100 ml needed in tray) - 0.2% Ponceau S stain - 3% Trichloroacetic acid - 3% Sulfosalicylic acid c. Rinse solution 5% glacial acetic acid d. Dehydration soln full strength ethyl alcohol e. Clearing soln 30% rgt-grade cyclohexanone in denatured alco. Procedure: A. Electrophoretic Separation 1. Prepare cells & membrane for sample application a. Fill reservoir w/ buffer soln. to fluid level b. Precondition a C-A membrane in buffer for 2 mins (forceps) c. Place membrane bet. 2 blotters & dry lightly d. Put membrane in bridge accdg. to reference hole index 1st sample application pt. e. Place bridge w/ mounted membrane onto cell (membrane ends should be submerged in buffer); reference hole in LL f. Place cell cover & lid 2. Sample application ensures that shape & size of all sample applications are uniform as possible a. Drop of sample on parafilm & cover w/ small beaker b. Sample picked up & deposited by ribbons on white button ribbons pick up 0.25 L by capillary action c. Retract using red button d. Put applicator on cell (rider in last sample positioning groove to the right); touch white button (tip strikes membrane) e. 5-7 sec. then press red button to retract tip 3. Electrophoretic separation function switch of Duostat at 0300 (constant voltage mode) & voltmeter switch to 0-500 volts
a. b.
Turn output adjust switch; adjust to 25o volts
Measure starting current: ammeter switch to 0-15 milliamp; Total starting current: bet. 8 & 12 milliamps c. Electrophorese for 20 mins 17 mm separation of proteins d. Albumin fastest moving (toward anode), globulin slowest e. Red Duostat lead: anode on R; Black: anode on L f. Measure ending current (usually 12-14 ma) B. Color Development Separate protein fraction 1. Protein stain 7-10 mins (Ponceau-S fixative dye) 2. Membrane rinse 3 times in successive trays of 5% acetic acid; remove excess dye 3. Membrane dehydration 95% denatured Eth Alc; 1 minute; remove excess moisture; if inadequate: membrane softens 4. Membrane clearing 1 min.; glass plate support the membrane, at bottom of last tray 5. Hold membrane & plate at slight angle w/ 1 end on a pad of absorbent tissue; squeegee gently Too much pressure distort serum pattern Dry 10-15 mins in 100C oven Complete drying no smell of cyclohexanone 6. Put membrane (electrophoretogram) in plastic storage envelope C. Quantitation of Electrophoretic Separation a. Equipment & materials Beckman & Analytrol densitometer Membrane carriage Chart showing Analytrol tracing b. Procedure: 1. Mark limits of each component by: a) Direct reference to the strip b) Normal distribution curve c) Lowest point bet. peaks 2. Count the saw teeth each is 0.1 cm2; grp of 10 = 1 cm2 3. Compute for the total area 4. Compute for the individual percentages 5. Compute for the protein conc. (from refractometer reading) NOTES 1. Sample preparation - Specimen of choice: serum from blood thru venipuncture - Complete clotting coz fibrinogen in serum fraction bet. and globulin regions - Capillary tubes no heparin alters EP mobility of lipoP 2. Problems / troubleshooting a. CA membrane Trapping of air bubbles regions of non-conductivity (absence of buffer) irregular separation patterns White spots if blotted too dry irregular paterns Too little blotting smeared patterns Too much time spent in transferring membrane drying Bridge-mounted membrane w/c is too loose / sags irregular pattern (incomplete contact of application) Tearing too much spring tension b. Sample application Too much sample smearing & non-linear staining Too little sample too faint pattern / absent 1 globulin Incomplete coagulation - fibrinogen as artifact in region Contaminated applicator irregular patterns c. Electrophoretic condition Inc. ionic strength of buffer dec. migration length, more sharply define fractions, inc. current level, heat build-up
Dec. ionic strength inc. migration length, more diffuse
fractions, lower current levels & less heat Starting current troubleshooting guide 1) Meter pointer offscale to right short circuit bet reservoir 2) Meter above S.B. ma buffer level too high or conc, salts formed in corners of center partition (put silicone grease) 3) No meter reading membrane ends not in buffer, Duostat inoperative, pins or connectors loose 4) Meter below 3.5 ma buffer level too low, diluted buffer, narrow membrane 5) Bent applicator wires poor sample application 3. Other considerations a. Staining time Too short light spot in albumin & 1 will not show b. Acetate acid rinse time extended to 2 hrs w/o damage Too short too highly colored background c. Alcohol rinse Too long makes membrane soft & difficult to handle Too short not dehydrate membrane sufficiently d. Clearing time Too long makes membrane soft & difficult to handle Too short cloudy background e. Oven drying Excessive curling, shrinkage, fading Too short incomplete clearing