- Cut PVDF membrane to the appropriate size, activate with absolute
methanol for 5 sec, and incubate in distilled water for 5 min. For
electroblotting, equilibrate in transfer buffer and follow the standard blotting
procedure to transfer the proteins to the membrane. For dot blotting, keep the
membrane wet until ready to use.
- After protein has been transferred to the membrane, wash again in absolute
methanol for a few seconds and allow to dry at room temperature for 30
min or more.
- Block in 30 mL of 1X Western buffer (containing 0.1% Tween-20 and 0.2%
I-Block), gently rocking for 1 hr at room temperature.
- Add appropriate dilution of primary antibody (typically 1:5000 or 1:10,000)
prepared in 1X Western buffer (containing 0.1% Tween-20 and 0.2%
I-Block), incubate 30 min at room temperature, gently rocking. Wash 3 times in 20 mL 1X Western buffer (containing 0.1% Tween-20 and
0.2% I-Block) for 5 min each. Add appropriate dilution of secondary
antibody conjugated to alkaline phosphatase prepared in 1X Western buffer
(containing 0.1% Tween-20 and 0.2% I-Block), gently rocking for 30 min at
- Wash as in step 5. Additionally, wash twice with 1X Western buffer
- At the end of the second final wash, leave some buffer in the container to
keep the membrane moist. With the membrane facing protein side-up, add
0.5 mL of substrate solution directly into the remaining liquid, mix well,
and pipette (with a p10000) the solution over the membrane to ensure the
entire surface comes into contact with the substrate. Gently agitate for a
few minutes, remove the membrane to a paper towel, and let it dry
completely. The substrate solution can be reused immediately for additional
- Scan membrane using the molecular dynamics storm.
Western Blotting Solutions
- 10X Transfer buffer: 240 mM Tris, pH = 8.0; 1.92 M Glycine. Use 0.5X for
transfer in 20% methanol.
- 10X Western buffer: 200 mM Tris pH = 7.5; 1.5 M NaCl. To prepare 1X
Western Buffer, dilute 10X buffer to 1X, adding Tween-20 to 0.1%. Remove
50 mL and set aside for the last 2 washes. To the remainder, add I-Block
to 0.2%, heating gently with constant stirring until dissolved. Bring to
room temperature before using (containing 0.1% Tween-20 and 0.2%
- Primary antibody: For his tagged proteins - Anti-His monoclonal antibody.
- Secondary antibody: Goat anti-mouse alkaline phosphatase conjugated -
- Substrate: ECF chemifluorescent substrate—Mix substrate with accompanying
buffer as per manufacturer’s recommended instructions, prepare 1-mL
aliquots, and store at –20°;C.