3/16/201214

NuPage Gel Electrophoresis and Western Blotting

Modified Feb 9, 2009 I. Sample Preparation 1. Thaw cell lysates from 80 C freezer on ice. 2. Calculate dilution of samples based upon gel size and sample required. Use Excel spreadsheet from protein assay. 3. Vortex sample to mix. 4. Heat samples at 95 C for 10 mins. Use heating block on bench by window or adjustable heater/shaker on Shannons bench. Make sure it is set to 95 C in advance. 5. Spin samples briefly to collect at bottom of tube. 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. Fill the wells with samples: *Use special pipette tips for easier loading and make sure to get pipettes that match tips. - Add 3 uL MW markers to 2 or more wells. Use the Kaleidascope or 2-stripe markers and then RECORD in your notebook which marker is where. - Fill remaining 8 wells with 15 20 ul of sample. The volume depends on which gel you are using. - Fill each well slowly. Try to avoid bubbles in wells that may cause overflow into other wells - Keep track of which gel is which by using different patterns of MW marker. Match the + and -electrode ends (same colors) and set the apparatus to run for 120 min @ 125V.

4.

5.

3/16/201214

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 on shelves in back of lab. b. Make tray of TB for membranes, filters and pads c. Obtain green plastic pusher from gel supply area by window d. Add TB to plastic case for gel Soak pads and filter papers in TB. Unlock wedge and remove one gel at a time. Use gel knife to crack open plates. Insert knife into plate gap and separate. Repeat on all sides. Once open, use gel knife to cut out gel and remove excess gel at bottom. Use knife and green pusher to remove gel into case with TB. Put two pads inside blot module (4 pads for only 1 gel). Add filter paper and gel to top of pads. Then, add membrane, filter paper, and one more pad.

7. 8. 9.
10.

11. 12. 13.

14. 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)
15.

Squeeze blot module closed, place inside cell, and use tension wedge to lock in place. There should be ~1cm gap at top. Fill inside of module with TB + antioxidant (make sure it does NOT leak). Do not fill all the way to the top. Then, fill the remainder of the cell with MilliQ water ~ 600 mL (~2cm from top). Set up transfer apparatus at room temperature to run for 1 hour @ 30V.

16.

17.

18.

3/16/201214

II. Western Blotting 1x TBS-T dilute 20X stock Store on bench at room T Block (match to dilution of primary antibody) 50% OBB in PBS Primary Abs Dilute in Block buffer Most Abs 1:1000 dilution: Add10 L Ab per 10 mL Soln Secondary Abs 1:15000 dilution of antibody in Block: 0.67 L Ab per 10 mL Block. 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.
1. 2.

agitation.
3.

Place membrane in 10 mL block for 1 hr @ RT with constant

Place membrane in primary solution overnight @ 4 C with constant agitation or alternatively @ RT for 1 hr with constant agitation 4.
5. 6.

Pour primary solution back into its original test tube for reuse. Wash blot 5 X 7 min in TBS-T.

In the meantime, prepare secondary antibody solution at 1:15000 dilution of antibody in Block (10mL for one blot). Add 0.67 L Ab per 10 mL Block.
7. 8. 9.

Place membrane in secondary solution @ RT for 1 hr. Cover w/ Aluminum Foil. Wash blot 3 X 7 min in TBS-T. Develop blot using Licor. place blot protein side down If needed for later use, store the blot in Saran wrap with TBS-T at 4 C (will keep ~ 1

10.

month) III. Stripping & Reprobing Stripping Buffer 0.2N NaOH

1. Stripping a. Wash blot w/ dH2O for 5 min and shake b. Add ~25 mL of stripping buffer for 5 min and shake c. Wash blot w/ dH2O for 5 min and shake 2. Block again for 1 hr @ RT w/ constant agitation.(Repeat from step 2 from above)