a large weigh boat, wet Immobilon membrane (2 cm x 7 cm) in methanol, decant
methanol and wash briefly in standard TBST buffer.
12.5 ml blocking buffer, and incubate ≥1
hours at room temperature with gentle shaking (~45 rpm) to block membrane.
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.
with parafilm and incubate overnight at room temperature with gentle shaking
the antibody and do subsequent washes with TBST for 2X –5 minutes and
3X-10 minutes with gentle shaking.
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.
90 minutes at room temperature with gentle shaking.
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.
Pico chemiluminescence substrate by adding 2 ml Reagent A and 2 ml Reagent
B, mix and add to clean weigh boat.
the membranes in the chemiluminescence substrate and incubate 5 minutes at
room temperature with rapid shaking (~200 rpm).
the membranes and place between clear plastic sheets with the protein side
a paper towel to squeeze out excess liquid and dry outside of plastic
to expose for 1, 2, 4, and 8 minutes on film.
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.
X Standard TBST Buffer
benzethonium chloride 0.10 g
pH to 7.4
Bovine serum albumin
Newborn calf serum
1X Standard TBST buffer to 50 ml
Millipore ImmobilonTM -P Transfer Membranes
Cat. No. IPVH00010
290 Concord Road
Billerica, MA 01821
Affinity Purified Antibody Peroxidase Labeled Rabbit Anti-Goat IgG (H+L)
Cat. No. 14-13-06
910 Clopper Road
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