To prepare for Protein samples

1. Centrifuge around 5million cells(Volume doesn't matter) for 2,500x, 10 min.

2. Discard the supernatant with Vacuum chamber.

3. Mix the cell pellet with 1X PBS and centrifuge it for 2,500x, 10 min.

4. Discard the supernatant with Vacuum chamber.

5. Necessary amount of lysis buffer depends on the pellet size - for 20mg pellet -> 150micro liters of IP lysis buffer.

6. Put the lysated sample in an ice box for 10 min - flip it off every 3 mins.

7. Add up 3x SDS lysis buffer (Here I put 75micro liters because the IP lysis buffer amount was 150micro liters)

8. Put the sample in 95 degree of heater to denature proteins for 5 mins.

To prepare loading samples

1. Volume for 7 micro liter of ladder is enough.

2. Make the sample size up to 50 micro liters.

Ex) 30microliters of cell lysis, 1microliter of VP16 (10microgram per 1microliter concentration), 19 microliters of SDS lysis buffer = 50 microliters of sample is ready.

3. Load the samples.

I. SDS Gel electrophoresis

1. Prepare the separating gel and stacking gel solution without APS and TEMED.

2. If the SDS kit is ready to work, add APS and TEMED into the separating gel solution and pour a little bit of DW on top of the solution.

3. After 20~30 mins, take out the DW and check the gel is solidified or not.

4. Put TEMED into the stacking gel solution and pour it on the solidified separating gel.

5. Insert a stack carefully and leave it for 20~30 mins.

6. Take out the stack and fill up SDS loading buffer in the kit.

7. Load the samples.

8. Add more loading buffer and set up the machine with 80volts for 1.5 hours.

II. Membrane transfer

1. Prepare a membrane transfer kit.

2. Take out the gel from the SDS kit and put it on the membrane paper.

3. From the bottom to top, put in order of 1) Sponge - 2) Blot paper - 3) Membrane - 4) Gel (Now pour some M-transfer buffer to make it fully wet) - 5) Blot paper again - 6) Sponge again.

4. Close the kit and set up the voltage with 20 for 30mins ~ 1 hour.

5. Discard the gel and put the membrane in 5% Skim milk with 30ml of TBST buffer(1.5g of skim milk power for 5% here) for an hour.

III. Antibody tagging

1. Leave 5ml of the 5% skim milk buffer and mix it up with 1:2000 or 1:1000 of primary antibody(5micro liters of antibody : 5ml of buffer = 1: 1000)

2. Overnight it at 4 degree.

3. Wash it out with 1X TBST for 5 times for 5 mins each.

4. With 5% skim milk buffer, use 1:2000 dilution of secondary antibody (we have goat anti rabbit antibody) and leave it in room temperature for 2 hours.

5. Wash it out with 1X TBST for 5 times for 5mins each.

6. Check the protein level in the dark room.