BIO430 Lab Exercise: Protein Analysis
Protocol 1: Protein Extraction
Material
1.5ml microcentrifuge tubes
Fish muscle samples
Laemmli sample buffer (SB)
Knife
Quantity
10
5 (one of each)
1.5ml
1
1. Label two 1.5ml microcentrifuge tubes for each of five different fish samples.
2. Add 250l of Laemmli sample buffer to each of the fliptop microcentrifuge tubes (not
the screwcap tubes).
3. Cut a piece of each fish muscle about 0.25cm3 and transfer each piece into the
appropriately labeled microcentrifuge tube.
4. Close the lids on the tubes and flick the tubes several times (approximately 15 times).
5. Incubate samples for 5 minutes at room temperature.
6. Carefully transfer the buffer from each microcentrifuge tube into the appropriately
labeled (clean) microcentrifuge tube. Do not transfer the muscle sample.
7. Keep protein extractions at room temperature while you prepare your gels.
Protocol 2: Protein Electrophoresis
Material
Fish protein extracts
Actin and myosin standard
Prestained Kaleidoscope standards
15% precast polyacrylamide gel
Gel loading pipette tips
Electrophoresis module (gel box)
1X Tris-glycine-SDS running buffer (TGS)
Quantity
5 species
10l
6l
1
7 tips
1
500ml per gel box
1. Set up electrophoresis module as outlined in Appendix A. Two groups will share one
electrophoresis module.
2. Heat protein extractions and actin/myosin standard at 95oC for 5 minutes.
3. Load the polyacrylamide gels as outlined below. Group 1 gel will be stained and the
Group II gel will be utilized in the immunoblot (Western blot) assay.
Lane
1&2
3
4
5
6
7
8
9
10
Group 1
Volume
Empty
5l
10l
10l
10l
10l
10l
10l
Empty
Group II
Volume
Empty
5l
5l
5l
5l
5l
5l
5l
Empty
Sample
Empty
Prestained Kaleidoscope standards
Protein extraction #1
Protein extraction #2
Protein extraction #3
Protein extraction #4
Protein extraction #5
Actin/myosin standard
Empty
4. Electrophorese for 30minutes at 200V in 1X TGS buffer.
5. After electrophoresis, remove the Group I gel from the cassette and transfer the gel
into a container with 25ml Coomassie blue stain. Stain for ~1hr with gentle shaking.
6. Destain the gels in water with shaking, overnight.
Protocol 3: Western blotting
Material
1X Tris-glycine blotting running buffer
Blotting paper
Nitrocellulose membrane
Western blotting electrophoresis module
Fiber blotting pads
Frozen ice-pack
Test tube
Quantity
500ml
2
1
1
2
1
1
1. Using a ruler, cut off the top (stacking portion) of the gel.
2. Equilibrate the gel in blotting buffer for 15 minutes, with gentle shaking.
3. Label the edge of the nitrocellulose membrane.
4. Prepare the blotting “sandwich”:
a. Add blotting buffer to approximately 1cm depth in a container (glass dish).
b. Open plastic cassette inside the glass dish so that the black side is in the buffer.
c. Lay a wet fiber bad on the black side of the cassette.
d. Lay one wet blotting paper on the fiber pad.
e. Lay gel squarely on blotting paper and roll out air bubbles with test tube.
f. Lay wet nitrocellulose membrane on the gel and roll out air bubbles.
g. Lay one wet blotting paper on the membrane.
h. Lay a wet fiber pad on top of the blotting paper.
i. Close the cassette and clamp together with the white clip.
5. Set up the transfer module with the clack side of the cassette next to the black side of
the transfer module. Add the frozen ice-pack and fill with clotting buffer up to the white
clip.
6. Place lid on tank and blot at 20V for 2.5 hours.
Protocol 4: Immunodetection
Material
Anti-myosin primary antibody
Anti-mouse HRP secondary antibody
HRP color detection substrate
Wash buffer
Distilled water
Quantity
10ml
10ml
10ml
200ml
100ml
1. Membranes will be placed in blocking solution for a minimum of 2 hours on a rocking
platform.
2. Discard blocking solution and incubate membrane with 10ml of primary antibody for
20 minutes on rocking platform.
3. Rinse the membrane in wash buffer, 2X for 5 minutes each.
4. Discard the wash and incubate membrane with 10ml of secondary antibody for 20
minutes on rocking platform.
5. Rinse the membrane in was buffer, 2X for 5 minutes each.
6. Discard the wash and add 10ml of HRP color detection reagent, incubate for 10-30
minutes on rocking platform, watching for color development.
7. Rinse the membrane in distilled water, 2X and blot dry with a paper towel.
8. Air dry and cover with plastic wrap.
Appendix A:
Mini-PROTEAN 3 Gel Electrophoresis System: Components
Ready Gel Precast Gels
1. Ready Gel Cassette Preparation
a. Remove the Ready Gel from the storage pouch.
b. Gently remove the comb and rinse the wells thoroughly with distilled water or
running buffer.
c. Cut along the dotted line at the bottom of the Ready Gel Cassette with a razor
blade.
d. Pull the clear tape at the bottom of the Ready Gel Cassette to expose the bottom
edge of the gel.
Mini-PROTEAN 3 Electrophoresis Module Assembly and Sample Loading
1. Place a Gel Cassette Sandwich into the slots at the bottom of each side of the Electrode
Assembly. Be sure the Short Plate of the Gel Cassette Sandwich faces inward toward the
notches of the U-shaped gaskets (see Figure 1a).
Fig 1a. Place Gel Cassette Sandwich into the Electrode Assembly with the Short Plate facing inward.
2. Lift the Gel Cassette Sandwich into place against the green gaskets and slide into the
Clamping Frame (see Figure 1b).
Fig 1b. Slide Gel Cassette Sandwiches and Electrode Assembly into the clamping frame.
3. Press down on the Electrode Assembly while closing the two cam levers of the
Clamping Frame to form the Inner Chamber and to insure a proper seal of the short plate
against the notch on the U-shaped gasket. (see Figure 1c). Short plate must align with
notch in gasket.
Fig 1c. Press down on the Electrode Assembly while closing the two cam levers of the Clamping Frame.
Note: Gently pressing the top of the Electrode Assembly while closing the Clamping
Frame cams forces the top of the Short Plate on each Gel Cassette Sandwich to seat
against the rubber gasket properly and prevents leaking.
4. Lower the Inner Chamber Assembly into the Mini Tank. Fill the inner chamber with
~125 ml of running buffer until the level reaches halfway between the tops of the taller
and shorter glass plates of the Gel Cassettes.
Note: Do not overfill the Inner Chamber Assembly. Excess buffer will cause the
siphoning of buffer into the lower chamber which can result in buffer loss and
interruption of electrophoresis.
Fig 1d. Lower the Inner Chamber into the Mini Tank.
6. Add ~200 ml of running buffer to the Mini Tank (lower buffer chamber).
Mini Tank Assembly
1. Place the Lid on the Mini Tank. Make sure to align the color coded banana plugs and
jacks. The correct orientation is made by matching the jacks on the lid with the banana
plugs on the electrode assembly. A stop on the lid prevents incorrect orientation.
Power Conditions
1. Insert the electrical leads into a suitable power supply with the proper polarity.
2. Apply power to the Mini-PROTEAN 3 cell and begin electrophoresis; 200 volts
constant is recommended for SDS-PAGE and most native gel applications. Run time is
approximately 30 minutes at 200 volts for SDS-PAGE.
Gel Removal
1. After electrophoresis is complete, turn off the power supply and disconnect the
electrical leads.
2. Remove the tank lid and carefully lift out the Inner Chamber Assembly. Pour off and
discard the running buffer.
Note: Always pour off the buffer before opening the cams to avoid spilling the buffer.
3. Open the cams of the Clamping Frame. Pull the Electrode Assembly out of the
Clamping Frame and remove the Gel Cassette Sandwiches.
4. Remove the gels from the Gel Cassette Sandwich by gently separating the two plates
of thegel cassette. The green, wedgeshaped, plastic Gel Releaser may be used to help pry
the glass plates apart.
Note: To remove the gel from a Ready Gel Cassette, first slice the tape along the sides
of the Ready Gel Cassette where the inner glass plate meets the outer plastic plate.
5. Run the sharp edge of the Gel Releaser or a razor blade along each spacer to separate
the gel from the spacer. Remove the gel by floating it off the glass plate by inverting the
gel and plate under fixative or transfer solution, agitating gently until the gel separates
from the plate.
6. Rinse the Mini-PROTEAN 3 cell electrode assembly, Clamping Frame and Mini Tank
with distilled, deionized water after use.