Conduct SDS-PAGE according to standard procedures.
1. Cast gel and allow to polymerize
2. Prepare samples to be resolved
3.Load gel
4.Run gel (< 200 volts for ~ 1 – 1.5 hours)
Transferring proteins to nitrocellulose membrane.
Can either use “wet” or “semidry” apparatus.
Wet transfer:
1.Disassemble gel casting plates so the gel is flat on one side or the other.
2.Using a clean razor, remove the stacking gel and clip the top right corner of the blot (according to the loading).
3.Using gloved hands and tweezers, quickly cut a piece of nitrocellulose to the same size of the gel.
4.Pre-wet both the gel and nitrocellose in transblot* buffer
5.Cut four pieces of Whatman paper to the size of the nitrocellulose.
6.Assemble the ‘sandwich’ as below make sure the cassette is open with the black side down before assembly!
a.Wet and place a Dacron pad nearest the hinge
b.Wet two pieces of Whatman paper, place on the Dacron pad
c.Put gel right-side-up and back-to-front on the filter paper
d.Place pre-wet nitrocellulose on top of gel, matching the clipped edge of gel with the clipped edge of the nitrocellulose
e.Place two more pieces of wet whatman paper onto the filter
f.Wet and place the other Dacron pad on the top.
g.Close and lock the cassett, place it into the electrode cassette and cover with transblot* buffer.
h.Add a stir bar and ice pack, and start transfer
7. Time and voltage of transfer will vary depending on the properties of the protein and the transblot* buffer used.
Continuous Semidry Transfer:
1. Disassemble gel-casting plates so the gel is flat on one side or the other.
2. Using a clean razor, remove the stacking gel and clip the top right corner of the blot (according to the loading).
3. Using gloved hands and tweezers, quickly cut a piece of nitrocellulose to the same size of the gel. (measure the gel area in centimeters)
4. Pre-wet both the gel and nitrocellose in transblot** buffer
5. Cut 16 pieces of Munktell Grade 1F filter paper to the size of the nitrocellulose.
6. Assemble the apparatus as follows:
a. Soak 8 pieces of filter paper in trasblot** buffer and make sure they are air bubble free on the anode plate.
b. Place the pre-wet nitrocellulose filter onto the stack
c. Place the pre-soaked gel onto the nitrocellulose
d. Place 8 more soaked pieces of filter paper on the gel.
e. Purge the stack of any air bubbles with a glass pipette.
f. Assemble the cathode plate and start the transfer
7. The equation to calculate the amperage for a 1 hour transfer is:
a. 0.8 mA / cm2 gel.
8. After 1 hour, disassemble apparatus and western blot the membrane.
For discontinuous semidry transfer, see the LKB 2117 Manual for appropriate reagents and assembly conditions.
Western blotting.
1. Disassemble transfer apparatus and remove the filter (membrane) from the sandwich.
2. Rinse the filter twice for 5 minutes with TBS or TBS-0.1% Tween-20 [For most applications, TBS can be interchanged with PBS for all washes and incubations, pending you are consistent with one or the other throughout].
3. Block the filter in a solution of TBS-T, 5% non-fat dry powdered milk (w/v) for at least 1 hour at room temperature (or overnight at 4 degrees).
4. Add primary antibody to the blocked filter at the appropriate dilution and place in a seal-o-meal bag without air bubbles.
5. Incubate primary antibody the appropriate time and temp.
6. Wash the filter at least three times for five minutes each in TBS-T
7. Optional: Reblock the filter after primary antibody for 10-30 minutes.
8. Apply the appropriate HRP-conjugated secondary antibody (diluted in blocking solution) to the filter for at least 1 hour at room temperature.
9. Wash the filter at least three times in TBS-T (5 – 10 minutes each)
10. Wash the filter once in TBS.
11. Develop the blot using chemiluminescence (ECL) substrates.
Developing the blot using PIERCE Chemiluminescence substrates (Supersignal).
1. Mix equal parts of “stable peroxide solution” (white bottle) and “luminol solution” (brown bottle). For a minigel filter, 5 ml of each is ample in a square petri dish.
2. Mix by swirling and allow the solution to come to room temp (~ 5 minutes)
3. Incubate washed filter with developing solution for 1 minute, swirling constantly and keeping filter covered with solution.
4. Optional: Rinse filter with TBS
5. Place filter face down on a piece of saran wrap, and fold edges under to seal the filter
6. Place the wrapped filter into an X-ray film cassette and tape edges to hold in place.
7. In the dark room, expose film to the filter and develop using the automatic film processor.
8. In ambient light, mark the position of the mw markers on the autorad using a marker. The tape marks on the autorad can be used to align the filter with the autorad.
