Western Immunoblotting Standard Protocol:

Transcription

1 Gel Preparation: Clean plates, combs and casting frames and bottom strips with water and alcohol and allow to dry Place a short plate on top of the spacer plate Slide the two plates into the casting frame, keeping the short plate facing front Even two plates on a level count top Lock the pressure cams to secure the glass plates Put gray gasket on the bottom of casting stand Engage the spring loaded lever on the casting frame Add alcohol to check if any leak Mix Separating Gel as outlined below: Stock Solutions 6% 8% 10% 12% 30% acrylamide/0.8% bisacrylamide 3 ml 4 ml 5 ml 6 ml 4X Tris-Cl/SDS, ph= ml 3.75 ml 3.75 ml 3.75 ml H 2 O 8.25 ml 7.25 ml 6.25 ml 5.25 ml 10% ammonium persulfate 50 µl 50 µl 50 µl 50 µl TEMED 10 µl 10 µl 10 µl 10 µl Note: The percentage of gel you pick is according to the size of interested protein (Please see the chart on the bottom of this section). 15 ml will make 3 minigels Add 10% AP and TEMED, then swirl gently Suck up carefully into syringe trying to avoid air bubbles Pour in gel with needle Overlay with 100 µl dd H20 or n-butanol Allow 1 hr to polymerize Wash 3 times with ddh2o to remove all traces of alcohol, then fill to top with ddh2o Page 1 of 6

2 Gels can be stored at this stage for several weeks if wrapped in wet paper towel inside a plastic bag at 4 0 C Make Stacking Gel as outlined below: Stock Solutions 3.75% 30% acrylamide/0.8% 0.65 ml bisacrylamide 4X Tris-Cl/SDS, ph= ml H 2 O 3.05 ml 10% ammonium 25 µl persulfate TEMED 5 µl Dry separating gel by pouring off H 2 O and drying with filter paper Put comb in gel apparatus and load syringe with stacking gel solution Lay stacking gel carefully over separating gel and allow to polymerize for 30 minutes Page 2 of 6

3 Samples Preparation and Electrophoresis: Thaw samples and dilute accordingly (usually load µg per lane), add 2x or 5x sample loading buffer (please note every kind of comb teeth has a max volume to be allow to load) Boil samples with loading buffer at 95C for 5 min (for cell protein extraction by using 1x SDS this step is not necessary) and keep the boiled samples on ice until loading Remove the gel cassette sandwich from the casting frame and place the gel cassette sandwiches into electrode assembly with the short plate facing inward Slide gel cassette sandwiches and electrode assembly into the clamping frame Press down the electrode assembly while closing the two cam levers of the clamping frame Put chamber into mini tank Fill both chambers and 1/3 of tank with running buffer Take out the comb Carefully clean the wells (Note: sometime having small piece of gel on the top of wells) Load samples and standard (5-10 µl), don t need boil the standard If need to stain a gel with comassie blue for protein determination, load a second gel Connect electrophoretic apparatus to power supply (black to black and red to red) and run the gel at 100 volts until the dye are just off gel (may take about 2 hours), ensure current is about 15 ma per gel Page 3 of 6

4 Protein Transfer: Remove gel, in transfer buffer, from plates and rinse twice with transfer buffer and leave gel in transfer buffer until transfer For 2 nd gel, please refer to section on comassie blue staining Cut 1 piece of nitrocellulose membrane and 2 pieces filter paper per gel Soak nitrocellulose membrane, filter paper, and 2 pieces of foam in transfer buffer for 10 minutes Place black part of cassette down and place the following on the cassette to form the gel sandwich: o 1 piece of foam o 1 filter paper o Gel o Membrane o 1 filter paper o 1 piece of foam Close cassette and lock into place Cover palladium anode. Place into apparatus with black to back and fill with transfer buffer --- the membrane will face the positive end (anode) and the gel will face the negative side (cathode). Run at 100 volts with current between 0.12 and 0.15 amps for 1 hour or 20 volts overnight with ice cooling unit in cold room (At this point, the membrane can be placed in TBS 0.1% overnight in refrigerator) Page 4 of 6

5 Blocking and Primary Antibody: Before blocking, stain membrane with 0.1% Panceace-S for mins to check transfer Block 1 hr at room temp or overnight at 4 C with 7% skim milk solution (3.75 g milk powder in 50 ml 1X-TBS-0.1% TWEEN) or 5% BSA Rinse 2x with TBS 0.1% TWEEN Bring antibodies to room temperature before use, dilute primary antibody at right concentration in 5% skim milk TBS- 0.1% Tween solution or 5% BSA For Bag Method: Heat the bag sealer up Place membrane with antibody dilution into a plastic bag and seal Incubate for 2 hrs at room temperature or overnight at 4C on a rocker For Multi-screener Method: Lay the membrane on gasket with the antigen side face up and center the membrane Place the sample template on top of the membrane using guide pins insure that the template properly align and finger tighten the four screws using a diagonal crossing pattern ad below: Tilt the multisreen apparatus toward you (~30 O ) Using loading tip, load 600 µl into bottom unmarked holes of the channels (slow, careful delivery of samples to avoid trapping bubbles) Incubate for 2 hrs at room temperature or overnight at 4C at flat position Page 5 of 6

6 Western Immunoblotting--Standard: Secondary Antibody: Pour off primary antibody (may save for future use) from bag or aspirate of primary antibody from multiscreen apparatus Wash for 15 minutes with TBS-TWEEN 0.1% and repeat for two 5 minute washes Add secondary antibody diluted with 5% milk in TBS - 0.1% TWEEN using bag method or multiscreen apparatus Incubate for 1 hr with rocking at room temperature Detection with Enhanced Chemiluminescence: Pour off the secondary antibody Wash 3x15 min with TBS-Tween 0.15% Mix 1 ml ECL1 and 1 ml ECL2 (Note: changing tips between ECL1 and ECL2) Agitate 1-2 min Put membrane in paper protection sheet Exposure to X-ray film in case or develop in Fluo-S Max multiimage, the duration is depend on abundance of interested protein Page 6 of 6