Protocol

 

"Results are quickly made, but publication quality pictures take time and patience."

 

WESTERN BLOT USING CHEMILUMINESCENCE

 

  1. Using a large weigh boat, wet Immobilon membrane (2 cm x 7 cm) in methanol, decant methanol and wash briefly in standard TBST buffer.

 

  1. Decant TBST buffer.

 

  1. Add 12.5 ml blocking buffer, and incubate ≥1 hours at room temperature with gentle shaking (~45 rpm) to block membrane.

 

  1. Add 125 ul 1% thimerosol to give 0.01% final concentration.  Make appropriate antibody dilution, typically 1:3,000 by adding 4.2 ul primary antibody directly to the blocking buffer.

 

  1. Cover with parafilm and incubate overnight at room temperature with gentle shaking (~45 rpm).

 

  1. Decant the antibody and do subsequent washes with TBST for 2X –5 minutes and 3X-10 minutes with gentle shaking. 

 

  1. Make appropriate secondary antibody dilution, typically 1:30,000, by adding 1 ul HRP-conjugated rabbit anti-goat IgG (KPL) secondary antibody to 300 ul standard TBST.  Add 125 ul of this dilution to 12.5 ml antibody dilution buffer.

 

  1. Incubate 90 minutes at room temperature with gentle shaking.

 

  1. Decant the antibody and do subsequent washes with wash buffer for 2X –5 minutes and 3X-10 minutes with gentle shaking.  Can store overnight in TBST wash buffer at 4º C. 

 

  1. Prepare Pico chemiluminescence substrate by adding 2 ml Reagent A and 2 ml Reagent B, mix and add to clean weigh boat.

 

  1. Place the membranes in the chemiluminescence substrate and incubate 5 minutes at room temperature with rapid shaking (~200 rpm).

 

  1. Remove the membranes and place between clear plastic sheets with the protein side up.

 

  1. Use a paper towel to squeeze out excess liquid and dry outside of plastic sheets.

 

  1. Proceed to expose for 1, 2, 4, and 8 minutes on film. 

 

  1. Develop film, fix, wash and dry.  Based on image, development times can be adjusted and/or chemiluminescence substrate (see note 2).

 

Note 1:  Optimizing the primary and secondary antibody dilutions is critical to a successful blot.

Note 2:  If additional sensitivity is needed after development, 1/40th volume each of Femto solutions A and B can added be added to the substrate in step 11 and then repeat the remaining steps.  The volume of Femto solutions can be increased until the desired sensitivity.  If one overshoots and the sensitivity become too high and produces too much background, the blot can be rinsed for 5 minutes in TBST and then reincubated in fresh chemiluminescence substrate.

 

WESTERN BLOT SOLUTIONS

  

10 X Standard TBST Buffer               1000  ml

 

            100 mM Tris-base                    12.11 g

            1.5 M NaCl                                 87.66 g

            0.5% MgCl                                   5.00 g

1% Tween 20                                 10 ml

0.01% benzethonium chloride   0.10 g

 

Adjust pH to 7.4

 

Blocking Buffer                                      50 ml

 

            2% Bovine serum albumin        1.00 g

            2% Newborn calf serum           1.00 ml

            1X Standard TBST buffer to        50 ml

 

MATERIALS

 

Millipore ImmobilonTM -P Transfer Membranes

    Cat. No.  IPVH00010

Millipore Corporation

290 Concord Road

Billerica, MA  01821

800-645-5476

www.millipore.com

 

Affinity Purified Antibody Peroxidase Labeled Rabbit Anti-Goat IgG (H+L)

    Cat. No. 14-13-06

KPL, Inc.

910 Clopper Road

Gaithersburg, MD  20878

800-638-3167

www.kpl.com

 

Pierce Super Signal® West Pico Chemiluminescent Substrate

   Cat. No. 37069

Pierce Super Signal® West Femto Maximum Sensitivity Substrate

   Cat. No.  34095

Pierce Biotechnology, Inc.

P.O. Box 117

Rockford, IL  61105

800-874-3723

www.piercenet.com