Western Blot Protocol

Material: 1.30% Acrylamide (at 4C) 2. 1.5M Tris.HCl (pH 8.8) 3. 1.0M Tris.HCl(pH 6.8) 4. 10% Ammonium Persulfate (made within two weeks, at 4 C) Note: The AP in brown bottle cannot make the gel solid; use those in the 15mL centrifuge tube. 5. 10%SDS 6. 5*SDS loading buffer 25mL of 1mol/L Tris.HCl (pH 6.8) 10g SDS 30mL Glycerol 250uL beta-Mercapitaethanol 0.02g Bromophenol Blue Add dH2O to 100mL 7. 1* electrophoresis buffer 80mL of 10*electrophoresis buffer + 720mL ddH2O Note: dH2O will make the buffer cloudy and lose the buffering function, so have to use Milli-Q water to dilute 10* electrophoresis here. And electrophoresis buffer should be used only once. 10* electrophoresis buffer 30.3g Tris base 144.2g Glycine 10g SDS Add 800mL dH2O to make total volume 1L 8. 1*transfer buffer 100mL of 10*transfer buffer + 100mL Methanol + 800mL H2O Note: Transfer buffer could be used for three times. 10*transfer Buffer 30.3 Tris base 144.2g Glycine Add 900mL dH2O to make total volume 1L 11. TBS/T 10mL 10*TBS +90mL H2O + 50uL Tween-20

10*TBS 200mL of 1mol/L Tris.HCl (pH8.0) 88g of NaCl Add H2O to make total volume to 1L

Procedure: 1. Take out protein samples from -20C. Thaw them at room temperature. 2. Take the cast stand and place the rubber bases (grey color) on them. 3. Place the 1.5mm glass plates with the green holder onto a casting stand. Note: Place the black clips to secure both sides of the plates carefully. 4. Prepare resolving gel: 1) 10ml of 10% resolving gel into a 50mL tube: H2O 4.0mL 30% Acrylamide 3.3mL 1.5M Tris-HCl (pH 8.8) 2.5mL 10% SDS 0.1mL 10% AP 0.1mL TEMED 4uL Note: The target protein size range of 10% Acrylamide gel is 14KD to 205kD. 2) After completely mix, pour the solution between the glass plates up to a little above the green line. Add water to remove the bottle. 3) Allow the gel to solidify to half an hour. 5. Stacking gel and sample preparation while waiting for the resolving gel to solidify: 1) 2mL of 5% stacking gel into a 15mL tube: H2O 1.4 mL 30% Acrylamide 0.33mL 1.0M Tris-HCl (pH 6.8) 0.25mL 10% SDS 0.02mL 10% AP 0.02mL Note: Add 2uL of TEMED later, so the gel will not be solid in the 15mL tube. 2) Sample preparation

Add certain volumes of protein samples to PCR tubes according to the concentrations of proteins. Add 1/3 volume of SDS loading buffer and mix. Put the PCR tubes into PCR machine, heating them at 95C for 10min. 3) Prepare 1*electrophoresis buffer 80mL of 10*electrophoresis buffer + 720mL ddH2O Note: dH2O will make the buffer cloudy and lose the buffering function, so have to use Milli-Q water to dilute 10* electrophoresis here. And electrophoresis buffer should be used only once. 4) The resolving gel should be solid at this time. The leftover in the 50mL tube can be used to check and a line between water and gel should also be seen clearly. Pour out the water and remove the water with filter paper. 5) Add 2uL of TEMED to the 15mL tub to make stacking gel. After mix thoroughly, pour the solution between the glass plates. Insert a comb between the glass plates. Make sure the size of the comb is right. Allow the stacking gel to solidify for 20 min. 6. Protein electrophoresis: 1) Place the glass plates and gel into the electrode stand. Place another pair of glass plates on the other side of the electrode stand. Note: Large glass plate facing outside and the small plate facing the stand. 2) Pour the electrophoresis buffer to the electrophoresis tank. 3) Remove the comb carefully from the glass plates. And wells are rinsed by flushing with running buffer using a pipette. 4) Load 2ul of molecular weight marker to the first lane using sequencing pipette tips. Then load the samples. 5) Close the unit; then connect it to the power supply. Electrophoresis samples are at 60V for 20min then 150V for 1.5hrs. Time can be changed to make the dye reaching the bottom of the gel. 6) Make up 1*transfer buffer then put into the cold room. 100mL of 10*transfer buffer + 100mL Methanol + 800mL H2O Note: Transfer buffer could be used for three times.

7. Transfer 1) At 5 min before finishing the electrophoresis, cut PVDF membrane and immerse it into methanol. After 1 min, put it to pre-cold transfer buffer. Cut 4 pieces of filter paper and 1 piece of weighing paper, immersing them into the transfer buffer as well.

2) Carefully take out the gel from the electrode. Wash the membrane with pre-cold transfer buffer, agitating for 15min. 3) Make a sandwich Open transfer cartridge Place the scotch pad then two pieces of filter paper then measuring paper then gel on the black side of the cartridge Carefully place the gel on the membrane then roll out the air bubble with a glass pipette Place the other two pieces of filter paper then scotch pad on top Roll air bubbles again with a glass pipette Close and seal cartridge 4) Place the cartridge in the transfer apparatus. Make sure the black side of the cartridge faces the black side of the apparatus. 5) Fill the apparatus with 1* transfer buffer until gel and membrane are covered 6) Put the apparatus into the cold room. Connect it to the power supply. Transfer at 250mA for 1.5hrs. 8. Western Immunoblotting 1) Blocking buffer (5% milk in TBS/T) When the transfer starts, make up TBS/T from 10*TBS. 20mL 10*TBS +180mL H2O + 100uL Tween-20 Put 1g fat-free milk powder into 20mL TBS/T Water bath can be used to dissolve the milk powder. But need to be cold before the blocking. 2) After transfer, take out the membrane from the cassette. Place the membrane into a container and blocking buffer (20mL) so that the membrane is fully immersed into the blocking buffer. Incubate for 1hr at room temperature. 3) Wash the membrane with TBS/T for 5 min. Dilute the primary antibodies into 5% milk powder in TBS/T. Incubate the membrane in the primary antibodies at 4C overnight. 4) Wash the membrane with TBS/T for three times. Incubate and agitate for 5 min at each time. Dilute the secondary antibodies into TBS/T. Incubate the membrane in the secondary antibodies at room temperature for 1hr. 5) Wash the membrane three times for 5min each wash in TBS. Note: TBS rather than TBS/T here because Tween 20 will react with the development reagent. 9. Development

1) Take out the luminal kit from 4C. Mix equal amount of reagent A and B into a 15mL tube or 2mL tube. Note: Change pipette tips when pipette reagent A and B. Once they are mixed in the bottle, the whole kit cannot work. 2) Remove the membrane from TBS and dry it by dipping one corner on the kimwipe. Put the membrane onto a container like a dish cover, the protein side facing up. Drip the mixed reagent onto the membrane; make sure it covers the whole membrane. 3) Incubate the membrane in the chemidoc hood for 5 min. Then put the membrane into a plastic wrap. Wrap carefully so no air bubbles are between the membrane and the wrap. 4) Open Quantitive One. Choose Chemi High Sensi, live acquire.