Immunoblotting of RASSLs

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Contents

Overview

List of reagents is not yet complete

Procedure

I. SDS-PAGE

1. Remove precast 10% Tris-Glycine gel (Novex #EC6075) from refrigerator and cool to room temperature.

2. Prepare controls (+/-), standards and adjust to the same volume as samples.

  • Standard:5 ml Kaleidoscope Prestained standard (BioRad #1610324)
  • 5 ng Flag-tagged protein
  • 40 ml 1x sample buffer

3. Gel assembly (Novex Apparatus: X Cell II Mini-Cell #EI9002): Remove precast gel from bag. Peel off tape on bottom of gel. Place gel into the apparatus. Remove comb. Fill the inside chamber with running buffer. Rinse wells with buffer using a pipet. Check and make sure that there is no leakage from the inside to the outside chamber. Fill the outside chamber with running buffer (can reuse running buffer from previous run) until the buffer covers the opening on the bottom of the gel.

4. Load 45 ml - 50 ml of each sample into well using flat loading tips. Fill blank wells with buffer to minimize curving of bands along the sides of the gel.

5. Run gel at 125 constant volts for 90 min or until blue dye front has exited gel.

6. While gel is running, prewet nitrocellulose, filter paper and blotting pads in blotting buffer.

II. Gel Transfer

1. Remove gel from apparatus. Using a razor blade, slice the lower opening of the gel and remove gel pieces in the slot. Pop open plastic plates with a knife. Remove the top portion (1 cm) of the gel.

2. Assemble gel transfer sandwich in the following position, starting from the bottom:

 (facing + electroce) top pad
   filter paper
   nitrocellulose
   gel
   filter paper
 (facing - electrode) bottom pad 

3. Fill the inside of the transfer chamber with blotting buffer and the outside with water.

4. Transfer for 1-2 hours at 30 constant volts (Novex apparatus)

5. After completing transfer, disassemble apparatus.

6. Place nitrocellulose in container with blocking solution (50 ml of 5% milk in TBS). Incubate for 0.5-3.0 hours at room temperature, with gentle shaking.

7. Discard blocking solution, rinse nitrocellullose 1x for 5 min in TBS.

8. Place nitrocellulose in container with M1 antibody directly conjugated to HRP (Chromaprobe Inc., custom conjugated M1-HRP). Incubate for 1-3 hours at room temperature, with gentle shaking. Cover container with foil.

Incubation mix:

  • 1 mM CaCl2 (20 ml 1 M CaCl2)
  • 1% BSA (2 ml 10% BSA)
  • 0.25 mg/ml M1-HRP (5 ml 1mg/ml M1-HRP)
  • in 1x TBS, 0.1% Tween-20 (18 ml 1x TBS, 0.1% Tween)

9. Rinse blot 1x with TBS-T Ca++ wash buffer. Then wash 2x, 15 min per wash. III. Developing Blot

1. Remove nitrocellulose from wash buffer and place on saran wrap.

2. Combine 4 ml of solution #1 with 4 ml of solution #2 (Amersham #RPN2106). Immediately pour mixture onto the surface of nitrocellulose. Allow reaction to go for 1 min (exactly) at room temperature.

3. Pour off developing mixture, enclose nitrocellulose in saran wrap and place a fluorescent marker next to nitrocellulose. Then, transfer this to a film cassette.

4. In dark room, place a piece of film on top of saran wrap. Expose for 1 min at room temperature and develop film. If signal is low, increase exposure time.

Running Buffer

  • 1x Tris/Glycine/SDS buffer (10x from Bio-Rad 161-0732)

Blotting buffer

  • 1x Blotting buffer (25x Tris-Glycine Transfer Buffer For Blotting, Novex #LC3675)
  • 20% Methanol

TBS

  • 50 mM Tris HCl, pH 7.4
  • 150 mM NaCl

TBS-T Ca++ Wash buffer

  • 1x TBS (50mM Tris-HCl, pH 7.4, 150 mM NaCl)
  • 0.1% Tween-20
  • 1 mM CaCl2 ==Notes==

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

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