ECL Western Blot
updated by Kate Shen
(For the PDF version, click here.)
NOTE: This protocol uses the ECL Plus Western Blotting Detection Reagent from Amersham.
Electrophoresis
a. Prepare 10%SDS Page gel.
b. 1) If the samples are from tissue directly, dilute 25µg/7.5µl with your 7.5µl extraction buffer + protease inhibitor (1:7 mix).(this volume is for mini gel with 15 well comb)
2) If the samples are from GST purified protein, 62.5ng are good amount of protein fortransfer and detection.
c. Rinse the well with 1XSDS gel running buffer in tank, then loading the samples.
d. Run the gel at 180V for 45 minutes.
Blotting
a. Cut off and trash the stacking gel.
b. Soak the resolving gel in 10mM CAPS/10% methanol buffer.
c. Soak the transfer membrane in methanol for 5 second, rinse it with dH2O, and then equilibrate the membrane in transfer buffer for at least 15 minutes.
d. Assemble the blotting sandwich in the following order: black portion of cassette, sponge, Whatman paper filter paper, PVDF membrane, Whatman paper filter paper, sponge, red/or white portion of cassette.
e. Clamp together; place in holder with black-to back, with icebox next to black.
f. Run at 70-90 for 30 to 60 minutes.
Blocking The Membrane
Block non-specific binding sites by immersing the membrane in blocking buffer (3% non-fat dried milk in 0.1%(v/v) Tween 20 in PBST for 1 hour at room temperature on an orbital shaker. (Over night in a cold room (or refrigerator) is good also).
Primary Antibody Incubation
a. Dilute the primary antibody in PBST.
b. Incubate the membrane in diluted primary antibody for one hour at room temperature on an orbital shaker. (Over night in a cold room (or refrigerator) is good also).
c. Briefly rinse the membrane with two changes of wash buffer 1X PBST and then wash the membrane in 4ml/cm² of 1X PBST for 15 minutes at room temperature on a orbital shaker.
d. Wash the membrane for 3X 5minutes with fresh changes of 1X PBST at room temperature.
Secondary antibody incubation:
a. Dilute the HRP labeled secondary antibody (1:15000) in 1%BBB buffer (1g BSA in 1XPBST).
b. Incubate the membrane in diluted secondary antibody for 1 hour at room temperature on an orbital shaker. (Over night in a cold room (or refrigerator) is good also).
c. Briefly rinse the membrane with two change of wash buffer 1x PBST and then wash the membrane in 4ml/cm² of 1X PBST for 15 minutes at room temperature on a orbital shaker.
d. Wash the membrane for 3X 5minutes with fresh changes of 1X PBST at room temperature.
Detection
a. Removes the detection reagents from 2-8 °C storage place allow to equilibrate to room temperature before opening.
b. Mix the detection solution A and B in a ratio of 40:1(for example: 2ml solution A+50µl solution B). Ensure the final volume of detection reagent required is 0.1ml/cm²)
c. Drain the excess wash buffer from the wash membranes and place protein side up in a container(wrap the container with foil)
d. Pipette the mixed detection reagent onto the membrane cover the container with foil and let the reaction goes for 2 minutes.
e. Drain off excess detection reagent by holding the membrane gently in a forceps and touching the edge against a piece of tissue. Place the blot protein side down on the screen of Typhoon. Make sure that there are not bubbles between. Make sure that the membrane will not be dried during the process. If the membrane is dry, the background will be stronger in the image.
Wash Buffers
| PBST (pH 7.5) | |
| 80 mM Na2HPO4, anhydrous |
11.5 g |
| 20 mM NaH2PO4 | 2.96 g |
| 100 mM NaCl | 5.84 g |
dilute the required amount of Tween 20 in the PBS.
a 0.1% Tween 20 concentration is suitable for most blotting application.
| TBST (pH 7.6) | |
| NaCl | 8g |
| 1 M Tris HCl, pH 7.6 | 20 mL |
dilute the required amount of Tween 20 in the PBS.
a 0.1% Tween 20 concentration is suitable for most blotting application.
| Transfer Buffer, 1 L | |
| 25 mM Tris | 3.03 mL |
| 192 mM glycine | 14.4 g |
| 20% methanol | 200 mL |
