Pam Kreeger
Modified January 23, 2006
I. Sample Preparation
1. Thaw cell lysates from –80 °C freezer on ice.
2. Dilute to give total protein desired using 4X Reducing Buffer and water. Make 30uL per lane, remembering that only 25uL will be loaded.
3. Make up MW marker (15uL of Kaleidoscope marker:5uL of 4X Reducing Buffer)
4. Vortex sample to mix.
5. Heat samples at 70 °C for 10 mins.Use heating block on bench by window.Make sure it is set to 70 °C in advance.
6. Spin samples.
II. NuPage Gels
Part A. Running Gel
Solutions: 1x Running Buffer (RB)
40 mL 20x MOPS RB
760 mL MilliQ water
1x Running Buffer + Antioxidant
200mL of 1X RB (above)
500 uL antioxidant
1.Remove precast gels from package (stored in cold room).
* For all samples, use 4-12 % Bis-Tris gels from Invitrogen.
Cut open and dump out liquid in sink.
Wash outside with MilliQ water.
Pull off white stripe at bottom.
2.Lower buffer core into cell and insert gel cassettes on both sides (make sure the shorter well side faces inward).Insert tension wedge and lock in place.
*If only running one gel, be sure to place buffer dam between the core and the tension wedge.
3.Fill the buffer core with 200 mL 1X RB + antioxidant.Make sure this section does NOT leak before adding liquid to the outside.Carefully remove the comb and pipet out any air bubbles. Then, fill outer part of case with 600 mL of RB.
4.Fill the wells with samples:
*Use special pipette tips for easier loading and make sure to get pipettes that match tips.
- Add 15 uL MW markers to 2 wells
- Fill remaining 8 wells with 25ul of prepared specific sample solution.
- Fill each well slowly. Try to avoid bubbles in wells that may cause overflow into other wells
- When working with 2 gel plates, make sure to label the outside of the container.
5.Match the + and -electrode ends (same colors) and set the apparatus to run for 50 min @ 200V.
- Turn on (be sure Range Select is OFF). Turn Range to 250V/250A, then hit Start.
- Wait, then adjust min/max to get correct voltage.
- When done, turn down voltage, turn Range to off, then turn off.
Part B. Transferring Proteins
Solutions:
1x Transfer Buffer (TB) + antioxidant
50 mL 20x TB
1 mL antioxidant
200 mL MeOH **100 mL for only 1 gel
749 mL MilliQ water**849 mL MilliQ water for 1 gel
6.Transfer set up:
a. Membranes can be found in drawer at Ale’s bench (filter-membrane-filter)
b. Make tray of MeOH, tray of milliQ water, and tray of TB for membranes
c. Obtain green plastic pusher from gel supply area by window
d. Add TB to plastic case for easy gel removal to filter paper
e. Fill 600 mL plastic beaker with TB for soak pads and membranes.
7.Soak pads and filter papers in TB.
8.Use tweezers to dip both sides of membrane in MeOH, water, and soak in TB.
9.Unlock wedge and remove one gel at a time.
10. Use gel knife to crack open plates.Insert knife into plate gap and separate.Repeat on all sides.
11. Once open, use gel knife to cut out gel, remove wells and excess gel at bottom.
12. Use knife and green pusher to remove gel into case with TB.Center gel on top of filter paper.
DO NOT LET SOAK IN TB FOR LONG
13. Put two pads inside blot module (4 pads for only 1 gel).Add filter paper and gel to top of pads.
14. Then, add membrane, filter paper, and one more pad.
15. Repeat steps 8-12 for second gel and this time add two pads on top.(Note: 5 pads total for 2 gels and 6 pads total for 1 gel)
16. Squeeze blot module closed, place inside cell, and use tension wedge to lock in place.There should be ~1cm gap at top.
17. Fill inside of module with TB + antioxidant (make sure it does NOT leak). Do not fill all the way to the top.
18. Then, fill the remainder of the cell with MilliQ water ~ 600 mL (~2cm from top).
19. Set up transfer apparatus at room temperature to run for 1 hour @ 30V.
II. Western Blotting
1x TBS-T
dilute 5X stock
Store on bench at room T
Block (match to dilution of primary antibody)
5% w/v Blotto 5% BSA in TBS-T
0.5g dry milk per 10mL 1x TBS-T 0.5 g BSA per 10 mL 1x TBS-T
Store at 4 °C Store at 4 °C
Primary Abs
Dilute in Blotto or 5%BSA/TBS-T
Most Abs 1:1000 dilution: Add10 mL Ab per 10 mL Soln
Secondary Abs
1:10000 dilution of antibody in Blotto: 1 mL Ab per 10 mL Blotto.
Detection Reagents
Use Western Lightning or Ecl Advance, stored in cold room
1mL reagent A + 1mL reagent B (or 1/2 soln for cut blots)
1. Remove membrane paper from transfer buffer apparatus. **Remember which side the gel proteins transferred onto. Cut as needed- to prevent blot from drying, do in shallow dish of transfer buffer. Add dot to the TOP LEFT corner of each section and mark the MW standards.
2. Place membrane in 20 mL block for 1 hr @ RT with constant agitation.
3. Place membrane in primary solution overnight @ 4 °C with constant agitation or alternatively @ RT for 1 hr with constant agitation
4. Pour primary solution back into its original test tube for reuse.
5. Wash blot 3 X 10 min in TBS-T.
6. In the meantime, prepare secondary antibody solution at 1:10000 dilution of antibody in Blotto (10mL for one blot). Add 1 mL Ab per 10 mL Blotto.
7. Place membrane in secondary solution @ RT for 1 hr.
8. Wash blot 3 X 10 min in TBS-T.
9. Get supplies: saran wrap, detection reagent, pipette " tips, 15 mL tube, tweezers, and timer.
10. Place blot on Saran wrap.Pipette above reagent onto blot, covering the entire protein surface. Let sit for 1min.
11. Develop blot using Kodak Imager.
- place blot protein side down
- Camera dial set to 2 to image a blot; can use 32 for marker
- Ideal temperature is -20oC; can track temperature in Capture window with Ctrl-T
- hit Capture (use Predict to get exposure time
- to Invert colors, use Image Display, then invert
- to remove speckles- in Image Display, choose Filter- Median (5x5)
- Angle button can straighten gel; use box tool to find lanes and select bands
12. If needed for later use, store the blot in Saran wrap with TBS-T at 4 °C (will keep ~ 1 month)
III. Stripping " Reprobing
Stripping Buffer
3mL 20% SDS (2%)
0.75mL 2.5M Tris, pH 6.8 (62.5mM)
0.21mL BME (d=1.114, 14.3M stock; 100mM)
26.04mL dH2O
Stripping
a.Place blot in ~25mL Stripping Buffer for 30min @ 50 °C in water bath in a SEALED container. Agitate every 10 min.
b. Rinse 3 times with dH2O into the waste jar.
c.Wash blot 3 X 5 min in dH2O
d. Wash blot 3 X 5 min in TBS-T.
Block again for 1 hr @ RT w/ constant agitation.…(Repeat from step 2 from above)
To clean container – carefully wash and soak with soapy water to remove traces of BME.
