Western Blot Protocol (EJ edit 6/15/15)

Solutions and Reagents:

1. Cell lysis buffer: RIPA buffer (Thermo Scientific#PI-89900). For 8 ml of buffer, add 1 tablet each of Protease inhibitor and Phosphatase inhibitor.

2. 2X Laemmli Sample Buffer (SDS loading buffer) (BioRad# 161-0737EDU)

3. 10X Tris/Glycine/SDS Electrophoresis (Running) Buffer (BioRad# 161-0732EDU): To prepare 1L 1X: add 100 ml 10X to 900 mL dH2O, mix.

4. 10X Tris/Glycine (Transfer) Buffer (BioRad# 161-0734EDU): To prepare 1L 1X Transfer Buffer: add 100 ml 10X Transfer buffer to 200 mL methanol, + 700 ml dH2O, mix.

5. 10X Tris Buffered Saline (TBS) (Bioexpress# J640-4L) with Tween 20 (Sigma# p-9416): To prepare 1L 1X TBST: add 50 ml 20X TBS to 950 ml dH2O, mix and add 1 ml Tween 20.

6. Prestained Protein Marker (BioRad# 61-0374)

7. Blotting PVDF membrane (Millipore# IPVH00010)

8. Blocking buffer: 1X TBST with 5% blocking reagent (Roche-applied-sceince# 11096176001); for 200 ml, add 7.5g powdered milk to 200 ml of 1X TBST.

9. Secondary Antibody: Goat anti-Rabbit IgG-HRP (BioRad# 172-1019) or Goat anti-Mouse IgG-HRP (BioRad# 172-1011)

10. Detection Reagent: SuperSignal West pico Substrate kit (Thermo Scientific# 34087)

11. Membrane Stripping Buffer: Restore Western Blot Stripping Buffer (Thermo Scientific# 21059) Lab made (Tris HCl 62.5 mM, 2% SDS) add 7ul/ml 2-ME.

Tissue Preparation:

1. Label 2 sets of eppendorf tubes with sample names.2. Obtain tissue samples from freezer and keep in dry ice.3. Weight the empty tubes and retake measurement with tissue samples (mg)

(Record). Put remaining sample back in freezer (-80oC).

4. Add 10 times RIPA (ul) (as lysis buffer) solution according to weight sample and homogenize samples by sonication or using a mortar and a pestle on ice.

5. Leave on ice for at least 30 min.6. Centrifuge in cold room or 4oC centrifuge @ max. speed for 15 min.7. Transfer supernatant to new-labeled tubes.8. Dilute samples 1:1 into 2X Laemmli Sample Buffer (add 10 ul loading buffer to

10 ul sample) in labeled PCR tubes.9. Heat samples at 95-100oC for 10 minutes (boiling denatures proteins) in PCR

machine (protocol: PROTEIN). Microcentrifuge and keep on ice.10. Store the aliquots in -80oC.

Running Electrophoresis Gel

Loading Samples

1. Prepare running buffer.2. Insert SDS-PAGE gel (BioRad: Criterio TGX Precast# 567-1085). 3. Load 5 ul of sample into each well saving the first and the last well for prestained

molecular weight markers (3 ul in the first and 7 ul in the last to keep track of gel orientation).

4. Cover chamber and apply a volt of max 70V for stacking gel then turn it up to 120V for separating gel.

Transfering Gel to PVDF membrane

1. Activate a PVDF membrane by immersing it in 100% methanol for one minute.2. Keep membrane, sponges, and filter papers immersed in 1X Transfer buffer

before use.3. Remove glass plates with gel from electrophoresis apparatus and rinse in tap

water.4. Wet fingers and separating plastic with transfer buffer and slowly break the

cassette apart.5. Assemble a sandwich in a Pyrex dish containing a transfer buffer in the following

manner:a. Open a western transfer cassette (which consists of two plastic plates and

two sponges) with a sponge on each side of cassette. (Black side on your right and white on left).

b. Place a piece of whatman paper on the right sponge.c. Place gel on top of the whatman paper. (Be sure the gel lies flat on the

whatman paper).d. Place the PVDF membrane on top of the gel and place a sheet of Whatman

on top of the membrane.e. Remove bubbles by smoothing with gloved fingers, being careful to keep

the membrane wet.

f. Finish by closing the second sponge and plastic piece over the “sandwich."

6. Immerse the assembly in a blot cell, making certain that the membrane side faces the positive electrode and the gel faces the negative electrode (black side of cassette facing black part of cell).

7. Put a stirrer and an ice pack in cell and take to cold room.8. Run at ~80V for about 4hrs or 30V overnight in the cold room. (Prepare 5% milk

solution using TBST while transfer runs).

Membrane Blocking and Incubation with Primary Antibody

1. Remove PDVF from transfer apparatus and place membrane immediately into a small tray.

2. Wash membrane with TBST for 5 min at room temperature.Optional: Soak membrane in Ponceau solution to see the band patterns. Rinse with TBST for 5 min.

3. Wrap membrane in clear plastic wrap and cut the appropriate band size. Label all membrane strips with corresponding Abs to be used and cut a small piece at the bottom to locate beginning.

4. Block by adding 10ml of milk to membrane in the tray (to prevent non-specific protein interactions between membrane and the antibody used for detection of the target protein).

5. Allow the blot to block from for an hour on a low speed shaker at room temperature.

6. Add primary antibody (diluted in blocking buffer) and incubate overnight at 4oC.

End of First Day

Second Day

Incubation with Secondary Antibody

1. Wash membrane 3X with TBST for 5mins each.2. Incubate membrane with species appropriate HRP-conjugated secondary at

1:5000 for phospho proteins and p120 and 1:10000 for total proteins.3. Incubate at room temperature for 1 hour.4. Wash 3X with TBST for 5 min each after secondary antibody incubation.

Protein Detection

1. Prepare fresh detection buffer (ECL reagent). Take 1:1 ratio from each bottle for total end volume.

2. Cut segments of paraffin film and make dots using the ECL reagent. Put membrane on film with reagent face down and incubate for 3 min.

3. Drain membrane of excess solution (do not let dry).4. Get film cassette and wrap membrane with saran wrap and get it ready for

exposure to X-ray film. An initial 10 sec exposure should indicate proper exposure time.

Stripping

1. If membrane has been in wrap for a while, put in TBST for 5 to 10mins on a shaker.

2. Wash with 10ml stripping buffer for 30 min to 1 hr in incubating shaker at low speed (~70) at 60oC.

3. Wash with TBST 3X for 5 minutes each.4. Block with 10ml of milk for an hour at room temperature over low speed shaking.5. Go back to Incubation with Primary Antibody and follow the steps from there.