SDS PAGE & Western Blotting

Monday, May 15, 2017 3:09 PM

Method description:
Analysis of protein samples by size using reducing gel electrophoresis (SDS PAGE) and by immunoblotting using anti-His antibody.

Materials needed:
• Glass plates & combs (A & C)
• Casting stands & frames (B)
• Buffer tank (D)
• Running & transfer electrode modules
• Running buffer & transfer buffer (cold)
• Gel ingredients
• Protein samples
• 4x loading dye & protein ladder

Protocol:
1. Gel preparation: Recipes:
Running gel Running buffer, 10x (pH 8.3): Transfer buffer, 10x:
a. Clean glass plates and assemble with frames in casting stand. SDS 10.0g Tris 30g
b. Prepare resolving gel (only add APS & TEMED just before pouring). TRIS 30.0g Glycine 144g
c. Pour gel between glass plates (micropipette, macropipette or freehand) Glycine 144.0g to 1L with ddH2O
100ml 10x TfB
d. Leave ~2cm from the top of the plates, or 0.5cm more than comb width. to 1L with ddH2O Make up for 1x: 200ml MeOH
e. Immediately add a 1ml layer 0.1% SDS or abs MeOH on top. Dilute 1/10 for 1x 700ml ddH2O
f. Leave to set for ~10min, check leftover gel in tube to judge.
g. Pour off & discard top layer of SDS/MeOH & rinse with dH2O from squeegy
h. Dry space between plates above resolving gel (gently, don't scratch gel). Resolving gel (pH8.8)*: Stacking gel (pH6.8)*:
3.33ml Acrylamide (30%, 37:1) 0.67ml Acrylamide (30%, 37:1)
3.75ml TRIS (1.0M, pH 8.8) 0.625ml TRIS (1.0M, pH 6.8)
Stacking gel
100ul SDS (10%) 50ul SDS (10%)
a. Fill to top of front plate using P1000 pipette 2.72ml ddH2O 3.6ml ddH2O
b. Immediately insert comb diagonally, avoid trapping bubbles. 100ul APS (10%) 50ul APS (10%)
c. Dab/dry overlfow gel & discard in biohazard waste. 10ul TEMED 5ul TEMED
d. Leave to set for 10-15min (check leftover gel to confirm) =10ml of 10% gel =5ml of 4% gel

2. Sample preparation:
a. Measure protein conc of each sample E.g. for 1µg protein load in 20µl (max well vol >20µl):
b. Look up well capacity for selected comb size: [Protein] Vol (prot) Vol (H2O) Vol (4xLD) Vol (final)
i. Max protein load (µg)
3.3µg/µl 0.3µlκ 14.7µl 5µl 20µl
ii. Max loading volume
c. Make up samples with same [protein] & sample volume (dilute with ddH2O to 0.4µg/µl 2.5µl 12.5µl 5µl 20µl
κFor ease of pipetting, dilute 1µl of protein sample in 9µl H2O (10x
3/4 of final vol) - see example on right.
dilution) & use 3µl in sample preparation. Adjust vol (H2O) in sample
d. Note: do not necessarily need molecular grade or nuclease free H2O.
prep accordingly.
e. Add 1/4 vol 4x loading dye (reducing agent & weighs sample into well)
f. Boil samples at 95°C for 5-10min

3. Run samples on gel:

a. Remove glass plates containing gel from casting stand.
b. Assemble into electrode module, facing inwards, ensuring proper sealsee 1-4.
c. Place assembly inside buffer tank5.
d. Pour running buffer into compartment of electrode module until overflow.
e. Removes combs gently and continue to add buffer to max mark.
f. Monitor for ~5min to see if there is leaking of buffer from central space.
g. Load protein ladder and samples into wells from left to right, using
micropipette & loading guide (optional).
h. Place lid on tank in colour-guided orientation (red-to-red, black-to-black)
i. Connect to PowerPac (also red-to-red, black-to-black).
j. Run at 80-120V for 60-120min (base on separation of marker)

4. Set up for transfer

a. Prepare large flat containerᵠ with ~2cm of cold transfer buffer.
b. Place the transfer sleeve open into the buffer with the black side down. ᵠIn CB lab we use large blue Qiagen
c. Soak one sponge in buffer & place flat on black side of sleeve. container for setting up transfer
sandwiches.
d. Place sheet of Whatman paper over sponge also on black side.
e. Prepare another sponge & Whatman sheet for white side.
f. Prepare membrane for transfer according manufacturer's instructions:

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 f. Prepare membrane for transfer according manufacturer's instructions:
i. Soak for 5min in MeOH
ii. Soak for 5min in ddH2O
iii. Leave to soak in transfer buffer until run is complete.

5. Western blot
a. Turn off PowerPac & remove gel from running tank.
b. Discard running buffer into chemical waste drum.
c. Carefully pry apart the front & back glass plate using green spatula
d. Cut off stacking gel using green spatula & discard in red bin.
e. Place gel in appropriate orientation on Whatman layer on black side.
f. Briefly submerge all layers in transfer buffer.
g. Align membrane on top of gel in appropriate orientation, again submerge.
h. Place 2nd soaked Whatman sheet on top of membrane, then sponge.
i. Close transfer sleeve & briefly submerge in buffer, before sealing.
j. Place assembly in red/black electrode module (black to black, run to red).
k. Place transfer electrode module in buffer tank.
l. Place ice pack in empty space next to transfer module.
m. Put lid on electrodes. Optional: place whole buffer tank in box of ice.
n. Transfer at 100V for 75-90min.

6. Clean up!
a. While transfer is going, clean all apparatus used for gel running.
b. If possible, dry & pack away neatly, or leave O/N & pack away ASAP!

7. Blocking
a. When transfer is done, disassemble transfer sandwich.
b. Place membrane in small plastic container of TBS/Tween to rinse.
c. Pour out PBS/Tween & add 4-5ml milk (or 5% BSA in TBS/Tween).
>24hrs
d. Incubate on rocker at RT for 60min

8. Clean up!
a. While membrane is blocking, clean all apparatus used for transfer.
b. If possible, dry & pack away neatly, or leave O/N & pack away ASAP! Cleaning WB apparatus:
• Wash used items immediately - don't leave for later
9. Primary antibody (risk of exposing lab-mates to hazardous substances)
a. Add 1/1000 dilution of 1°Ab (αHis-tag) to milk • Do not leave WB apparatus drying on sink for longer
b. Incubate O/N at 4°C or at RT on rocker than 24hrs, dry by hand if needed.
• Glass plates & combs are super fragile & must be
10. Secondary antibody dried/packed away same day (not O/N).
a. Next day, pour off milk with 1°Ab & rinse membrane briefly with TBS/Tween • Plz be gentle with the cables of the electrode lid -
the wires lose contact over time if roughly handled.
b. Perform first set of washes:
i. Add a splash of TBS/Tween to container, put on rocker
ii. Replace TBS/Tween to the following schedule: 2x 5min, 2x 10min
c. Add 4-5ml milk to container
d. Add 1/5000 dilution 2°Ab (eg. GαR) to milk
e. Incubate for 60min at RT
f. Perform second set of TBS/Tween washes as above.

11. Detection
a. Take the following to GelDoc:
i. Membrane, moistened with a tiny bit of TBS/Tween
ii. Detection reagent (ECL substrate)
iii. P1000 & clean small tube (1-2ml)
b. When ready, blot away all TBS/Tween with paper towel.
c. Mix ECL substrate & peroxide 1:1 in tube (white bottle & brown bottle).
d. Place membrane container on flat surface, or even put membrane in GelDoc.
e. Coat membrane dropwise with ECL mixture (cover evenly).
f. After 2-3min, view membrane inside dark GelDoc cabinet via camera
i. Open Magic software on GelDoc computer
ii. Click on camera in top left corner -> Open K12CH
iii. View -> Autoexposure (will run increasing exposure lengths until bands
detected, usually 7-10s)
iv. Change to Preview mode & set 7-10sec exposure time
v. As each preview runs, toggle focus until bands are clear
vi. Click File -> Capture
g. Export photos as images & email to yourself from GelDoc computer.

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Notes:
• Pouring & running gels requires the preparation of several buffers & stocks. However, once this has
been done, these stocks last for some time and make preparing the gel a much easier task.
• APS & TEMED are catalysts for polymerization of acrylamide -add only when about to pour gel, i.e.
wait until resolving gel is set & prepped, then add catalysts to stacking gel.
• Check that the buffer tank doesn't get too hot (electrolytes get low or voltage is too high).
• If gasket on running module has come out, ensure it is replaced in correct orientation (below).

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