Dept. of Molecular and Cell Biology

Western blotting

April in 2002

Kijeong Kim

Leighton Laboratory Dept. of Molecular and Cell Biology UC Berkeley SDS-PAGE

Transfer to blotting paper

Blocking

Incubation with primary Ab.

Incubation with secondary Ab.

Colorimetric Reaction

Materials  Prestained marker protein  Transfer buffer 20 mM Tris, 150 mM glycine, pH 8.3, 20% Methanol

Tris base 14.52 g 9.69 g 4.84 g 2.42 g Glycine 67.56 g 45.04 g 22.52 g 11.26 g Methanol 1.2 L 0.8 L 0.4 L 0.2 L H2O to 6.0 L to 4.0 L to 2.0 L to 1.0 L

 10X Tris-buffered saline (TBS) 200 mM Tris, 8.5% NaCl, pH 7.5

Tris base 24.2 g NaCl 85 g D.W. 1000 ml

 Tris-buffered saline with Tween-20 (TBST) Dilute 10X TBS with D.W. ten times. Add Tween-20 to be 0.1% or 0.3% finally.  Tris-buffered saline with Tween-20 and 0.5% Gelatin (TBSTG) Add 5 g into 1000 ml TBST Boil or exposure in microwave oven till dissolved and cool it.

 Nitrocellulose membrane 0.45 m pore size

 Ponceau S solution (for reversible staining) 0.5% Ponceau S 1% acetic acid Alternatively you can purchase it from sigma chemical company.

 Blocking solution 5% non-fat dried milk (skim milk)

Skim milk powder (Difco corp.) 1 g TBST or TBSTG 20 ml

 Primary Ab. Polyclonal or monoclonal Ab.  diluted in TBST, store frozen at -20C The diluted Ab. can be reused up to 10 times Dilution : Polyclonal Ab.  1:100 ~1:1000 or dilute depending on titre. Monoclonal Ab. ascitic fluid  > 1:1000

 Secondary Ab. Conjugated with AP (Alkaline phosphatase) Divide in 10 l aliquots store frozen at -20C Dilute 1:3000 with TBST, store frozen at –20C 1 aliquot in 30 ml TBST.

 AP substrate solution. Make immediately prior to development of Western blot.

 Washing solution Use TBST or TBSTG Prepare adequate volume. Procedure

* Wear gloves to protect the contamination of skin proteins * Use chilled transfer buffer at 4C.

1) Run SDS-PAGE with prestained molecular weight marker protein. 2) Isolate the gel 3) Mark the gel for orientation by cutting the upper left corner. Soak the gel in transfer buffer for 30 min. (Equilibration) 4) Cut NCM and 2 pieces of Whatman 3M filter paper to be the same as the gel. Pre-soak the NCM in transfer buffer for 5 min. 5) Rinse buffer chamber with D.W. 6) Fill a tray with transfer buffer to a depth of at least 1 inch. (The cassette should be loaded under buffer to avoid trapping air bubbles between the different layers.) * Should be no air bubbles between the layers (By rolling a pipette over the surface) i. Open the cassette by sliding ii. Place one half in the tray iii. Place one foam sponge pad on the cassette half Press on the sponge to force out the trapped air bubbles. iv. Place one piece of filter paper on the sponge. v. Place the transfer memb. (NCM) on top of the paper. vi. Place the gel on top of the memb. * Very important not to trap air bubbles between the memb. and the gel. vii. Cover the gel with filter paper. viii. Add a second foam sponge pad and press gently to force out trapped air. ix. Place the second cassette on the stack and lock the cassette gently. 7) The cassette with its packing should hold the gel in firm contact with the transfer memb. without squeezing the gel. If the packing seems loose, you may add another sheet of filter paper, If you think the packing is too tight, the upper sponge may be replaced with a second piece of filter paper. 8) Place a stirring bar in the bottom of the chamber to prevent buffer consumption on the electrode. Stirring speed should be moderately low speed. 9) Place heat exchanger If you use conventional larger Hoefer transfer chamber, you must use heat exchanger. If you use small Hoefer transfer chamber, you must insert the ice pack. If you use Bio-Rad Transfer chamber, it may not be required but in the cold room or cold chamber. 10) Fill the buffer tank with transfer buffer sufficient to cover the gel/memb. sandwich. 11) Insert the cassette into the blotting chamber. * Attention to the orientation : NCM should be between the anode (Red) and gel. If you use only on cassette, the cassette should be near anode. 12) Tap the chamber a few times to shake out air bubbles. 13) Cover the chamber with the power lid. 14) Run the electrotransfer in the constant voltage mode At 100 Volt , for 1 hour in the cold room or cold chamber. * The current will increase, take care not to allow the buffer to heat up beyond about 60C since the plastics will wrap. * For details, refer to the instruction manual of the model. 15) After completion, turn off the power, open the lid, lift up the cassette, open the cassette and separate the NCM from the gel. Discard the filter paper but you can reuse it later after dried at the clean area. 16) Transfer buffer can be reused several times but if the current increases highly, this means loss of buffer and it may cause heat. In this case use fresh buffer. 17) Staining of NCM Stain the memb. in Ponceau S solution for 1~5 min. Destain it in water for 1 ~ 2 min. Photograph the stained NCM. You can cut the NCM to prepare the strips. Completely destain the NCM with TBS or TBST for 10 min. 18) Blocking the membrane Place the membrane in the small plastic chamber or incubation tray containing blocking buffer (30 ml for the mini-gel size membrane or for incubation tray 1 ml in each slot) and shake or rock in the moderately speed for 1 hour. If you incubate it O/N at 4C, you have to add sodium azide of 0.1% final conc. 19) Incubation with primary antibody Discard the blocking solution and wash the chamber with TBST shortly several times. Fill it with 20~ 30 ml of appropriately diluted primary Ab. solution for the mini-gel size membrane or for incubation tray 1 ml in each slot Incubate for 1 hour at 37C or for 2 hours at RT. You can incubate it O/N at 4C, if the antibody titre is so low.

20) Washing Collect the primary Ab. solution and freeze at -20C. Fill the chamber or tray with TBST or TBSTG and shake for 5 min. Repeat four times replacing the buffer with new TBST or TBSTG. 21) Incubation with secondary antibody Replace the blocking buffer with appropriately diluted secondary Ab. solution. Incubate for 1 hour at 37C or for 2 hours at RT. 22) Washing Collect the primary Ab. solution and freeze at -20C. Fill the chamber or tray with TBST or TBSTG and shake for 5 min. Repeat four times replacing the buffer with new TBST or TBSTG. 23) Development For AP substrate Kit (Bio-Rad incorp.) Dilute 25X Substrate buffer with D.W. to make 1X working solution. Add reagent A 1/100 volume of the above solution and vortexing. Add reagent B 1/100 volume of the above solution and vortexing. Store the original reagent A and B again at -20C.

After discarding the washing buffer, add the substrate solution 20 ml for mini gel size membrane or 1 ml in each slot for incubation tray and shake it till the color is developed fully (5 ~ 10 min.)