WESTERN BLOT PROTOCOL

Day 1

Gel Preparation:

1.   Clean plates by spraying with 70% ethanol and drying with a towel

2.   Make plate sandwich and align in clamp (thumbnail shouldnŐt catch)

Effect of differing conceentrations of Acrylamide on protein separation


3.   Make separating gel cocktail containing the proper amounts of:

a.   Water (added first)

Acrylamide Concentration (%) 5 7.5 10 12.5 15
Linear range of Separation 50-157 36-94 24-75 18-68 12-43

b.   30% Acrylamide solution

c.    4 X Tris-SDS buffer

d.   10% ammonium persulfate

e.   TEMED

Final % Acrylamide 5% 7.5% 10% 12,50% 15%
Water 5.75 4.9 4 3.2 2.4
4X Tris-SDS (pH=8.8) 2.5 2.5. 2.5 2.5 2.5
30% Acrylamide 1.65 2.5 3.33 4.2 5
10% APS 100 100 100 100 100
Mix well          
TEMED 10 10 10 10 10

Add 3.4ml to each mini gel with 0.75mm spacers

4.   Overlay with 100µl of 70% Ethanol

5.   Allow to polymerize 20 minutes

6.   Aspirate the butanol and wash 5 X with water

7.   Make the 5% stacking gel

a.   4.67ml water

b.   2ml 4X Tris-SDS (pH=6.8)

c.    1.33ml 30% Acrylamide

d.   65µl 10% APS

8.   Take 1ml to wash the top of each of the gels

9.   Place the combs (15well or 10well depending on the number of samples) in the gel at an angle

10.        Add 10µl TEMED

11.        Pour 1ml (or enough to fill) at the corner under the comb

12.        Push the comb down at an angle making sure not to trap any bubbles.

13.        Allow the gel to polymerize for 20 minutes

Gel Run:

1.   Take out and defrost samples and prestained protein marker (from –20oC freezer).

2.   Put gels in the gel core unit so that the smaller plate is facing inward

3.   Press the gels down and then the core unit and close the clamps

4.   Pour 1X Running Buffer in until it reaches the level of the top of the gaskets.

5.   Remove the combs carefully and straight up as to not alter the lanes

6.   Rinse out the lanes with the 1X buffer.

7.   Add 4µl (10-well) or 2.5µl (15-well) STD and 2-20µl of sample to the lanes, vortexing before adding. Return standard and samples to freezer when finished.

8.   Now place the gel core into the gel tank.

9.   Fill 1X buffer up to halfway up the clamps on the front of the gel core

10.        Put on electrode top (make sure black-black and red-red).

11.        Using Power Pac 300, plug in electrodes (black-black and red-red)

12.        Run at constant voltage at 100V for 20min and 200V until front leaves the gel (~40 min).

Transfer:

1.   Approximately 5-10 minutes before the 1 hour for gel running is complete: Take out tub, four filter papers, two membranes, two black/white cartridges, tray and transfer Buffer.

2.   Label both membranes (donŐt touch w/ hands!) on upper left hand corner.

3.   Wet membranes in methanol

4.   Rinse 2X with water

5.   Place in transfer buffer on orbital

6.   Once gel is done running, turn off Power Pac 300, take out the gel core, dump the 1X Running Buffer

7.   Remove the gels from the core.

8.   Using the wonder wedge, separate the two plates from each other and press the gel down onto the smaller plate.

9.   After the 2 plates are separated and gel is on the smaller plate, cut off the lanes.

10.        Open 1 black/white cartridge and put in tray with transfer buffer.

11.        Lay first sponge on the black side of the cartridge.

12.        Lay one filter paper on the gel (on small plate), and transfer the gel to the filter paper using the wonder wedge.

13.        Lay this on the sponge (w/filter paper first on sponge).

14.        Lay membrane on the gel, face down (as it is sitting in the transfer buffer) and center first.

15.        Lay other filter paper on the membrane.

16.        Hold one hand on filter paper and roll out any bubbles with a pipette in the other hand.

17.        Lay other sponge on the filter paper.

18.        Close up the black/white cartridge and place in the black and red gel transfer unit, so that the black side of cartridge faces black connection of red and black gel transfer unit.

19.        Take 2nd gel from gel core unit and repeat steps 6-16 for other gel so that both black/white cartridges are in the black and red gel transfer unit.

20.        Place red and black gel transfer unit into the gel tank.

21.        Put in small stir bar and place an ice block behind black and red gel transfer unit.

22.        Fill gel tank to rim with Transfer Buffer, using all Transfer Buffer that is in tray and tub. Return extra Transfer Buffer to fridge.

23.        Put on top (make sure black-black and red-red).

24.        Place gel tank on stirring plate and stir at a high velocity.

25.        Using Power Pac 200, plug in electrodes (black-black and red-red), set constant voltage to 100V and run for 30 minutes. Amperage should be around 300 mA.

26.        While transferring, make 3% milk using 1.5g dry milk and 50ml TTBS.

Blocking:

1.   When transfer is finished, turn off Power Pac 200, take off the top.

2.   Return ice block (now liquid) to freezer.

3.   Take out black and red gel transfer unit and remove and open both cartridges

4.   Discard the filter papers and gel and place each membrane face up into a small container with 25 ml of milk solution.

5.   Rock slowly for 30-60 minutes.

6.   While rocking, return Transfer Buffer to the fridge, return the stir bar, rinse out the gel tank with water, rinse the sponges and dry flat, and rinse the red and black cartridge.

7.   After rocking for 30-60 minutes, pour the milk into a 50ml Falcon tube and rinse with TTBS (enough to cover membrane, approx. 6-10 ml).

8.   Dump out TTBS, add new TTBS, and shake for 5 minutes.

Primary Antibodies:

1.   Make primary antibody solutions (MILK: 3% milk in TTBS, BSA: 3% BSA in TTBS).

2.   Take membranes from small container, set on piece of paper and cut membranes according to molecular weight (in KDa).

3.   Put membrane, protein side up, into a 6-well plastic gel container filled with approx. 6-10 ml TTBS.

4.   Dump TTBS from 6-well plastic gel container and quickly add enough primary antibody to cover (6-10 ml) for each targeted protein.

5.   Rock slowly overnight at 4oC.


2nd Day

Washing with TTBS and Secondary Antibodies Phospho:

1.   Pipette out primary antibodies and return to respective tube and immediately put in TTBS.

2.   When all wells have been cleared of their primary antibodies and are filled with TTBS, dump out TTBS, and pour new TTBS in.

3.   Shake for 5 min, dump TTBS, then add new TTBS.

4.   Repeat step 3 twice more (for a total of 15 minutes of washing in TTBS).

5.   While washing, make secondary antibodies in 3% milk solution as before  and add appropriate secondary antibody (anti-rabbit, anti-mouse, or anti-goat) for each targeted protein.

6.   After the wash is complete, dump out TTBS from each well, and add 6-10 ml of secondary antibody and rock slowly for 1 hour.

7.   After 1 hour, dump secondary antibodies, and repeat steps 3-4 for a total of 15 minutes of washing with TTBS again.

Supersignal Reaction:

1.   Add 250 ml of each of two West Dura reagents per membrane to the plexiglass plate and mix.

2.   Lay the pieces of membrane protein side down on Kimwipe and blot dry.

3.   Lay all pieces of membrane protein side down on the West Dura reagents.

4.   Let sit for 5 minutes.

5.   After 5 minutes, take membranes and lay protein side down on Kimwipe

6.   Place in plastic bag.

7.   Close plastic bag with heat sealer.

8.   Tape to plate w/membrane protein side up.

Read on Computer:

1.   Open GeneSnap program.

2.   Make sure that it is set for Chemi

3.   Open door to machine, place the membranes on the transilluminator, and focus with the computer.

4.   Take an image of the membranes

5.   Press Auto Expose to determine the length of exposures required.

6.   Then manually expose so that the image is clearly visible.

7.   Adjust the exposure of the image in the histogram box by moving the red line until all bands are visible but not saturated.

8.   Export image to gene tools.

9.   Remove the membranes from the Chemidoc, slice the bag open, and put membranes, protein side up, in TTBS in 6 well container.

Stripping Membranes of Phospho Antibodies:

1.   When both the membranes have been read on the computer, and all pieces have been put back into TTBS, dump TTBS, and add approximately 6-10 ml Western Stripping Solution to each piece of membrane.

2.   Shake for 20 minutes.

3.   While shaking with Western Stripping solution, prepare 50ml 3% milk.

4.   Pipette Western Stripping solution from each well and put in fridge. Immediately put TTBS in. Dump TTBS, and then add new TTBS.

5.   Shake for 5 minutes. Dump TTBS, and then add new TTBS.

6.   Repeat step 4 for 5 minutes twice, for a total of 15 minutes of washing with TTBS. Leave membranes in TTBS.

7.   Block and reprobe with total antibodies as before