Western Blot Protocol:

1. Collect all items and clean plates with 70% ethanol

a. Use 1.5mm plate
2. Add them to the plastic set up, make sure theyre tight
3. Reheat cell lysate at 95oC for 5 mins
4. Using the minicentrifuge, minicentrifuge it for a little bit
5. Create the gel mixtures, add the ingredients in order theyre listed below use and label test
50ml tubes

2 x 6% 2 x 8%
H20 10.8 mL 9.4 mL
1.5 TRIS 5 mL 5 mL
30% BIS ACRYL 4 mL 5.4 mL
10% SDS 200L 200L
10% APS 100L 100L
TEMED 10L 10L

6. Place these solutions into the plastic set up, 9 mL of gel solution per plate
7. Add some methanol on top, let gel polymerize for 1HR
8. After gel has polymerized, pour out methanol
9. Prepare stacking gel solutions
4X STACKING GEL
Water 7.0 mL
0.5 M Tris 1.35 mL
30% Bis Acryl 1.75 mL
10% SDS 106.5 L
10% APS 106.5 L
TEMED 5 L

10. Add stacking gel solutions (5ml) and add combs (1.5 mm 15 well comb) let it polymerise for
another hour
11. Prepare transfer and running buffer, place transfer solution in glass jar and place in fridge
1L TRANSFER SOLN 1L RUNNING SOLN
100mL 10x transfer buffer 100ml 10x running buffer
100mL methanol 900 dH2O
800mL dH2O

12. Take out gels from plastic set up and move it to a new holder with a new set up:
pour running buffer

13. Remove combs and add ladder + cell lysates into gel and run it. Do it slowly to
make sure no bubbles form
14. Pour the rest of the running buffer inside
15. Run for 10 mins at 70 V and then 1hr at 150 V
 Transferring:

1. Soak PVDF membrane in methanol for 3 mins to activate it

2. Soak for a few minutes in transfer buffer use tweezer
a. Soak 2x sponge and 8x papers as well
3. Take gel out of glass and Cut the top part off gel, leaving only bottom part of gel place it in
transfer solution
4. Set up the holder as such
(-) Black holder
1 sponge
2 papers
GEL
Membrane
2 papers
1 sponge
(+) Clear holder
5. make sure there is no bubbles in the sandwich, if there is, use a broken pipette and roll out the
bubbles
6. place sandwiches on cubicle, black on black and clear on red, pour transfer buffer inside set up
and use curved lid
7. place icepack inside along with stir bar inside cubicle set up and then put inside cold room
8. spin magnet level 60-80 and then run for 100V for 2.5 hours, add ice on the outside and then
check every hour, and then change ice as well
9. clean everything up using hot water and distilled water

Blocking (3% MILK TBST Solution):

1. take the two small plates and fill them with TBST
2. place membrane in there
3. place on rocker, 80 RPM for faster washing
4. throw everything away except sponges + holders, rinse these and put them back
5. measure 2x 15ml tbst
6. use plastic plate for milk powder and measure 2x 0.45g milk powder
15mL TBST x 3% milk= 0.45g milk powder
7. remove the tbst
8. add the 15ml tbst
9. sprinkle milk powder on blot
10 Antibody solutions:
10. turn rocker (90 rpm) until milk is dissolved, slow down to (30 rpm)
11. incubate plates overnight on rocker BRAC1:
next day primary antibodies: 1 L: 1000 L
1. throw out milk solution GAPDH:
2. wash with TBST for 5 minutes (4 times) at 80 RPM
3. cut the blots in proper ranges 1 L: 10,000 L
4. get Ab from fridge, 1 L: 20,000 L
a. for brca1= rabbit Abs for GAPDH= mouse Abs
 5. place on rocker in cold room, 30-40 RPM for 2.5 hours

next steps:

1. drain out excess liquid and put it back in Ab conical tubes

2. put conical tubes back into fridge
3. put tbst into wells and wash on rocker at 90 RPM for 5 mins (4 X)

solution for attaching secondary antibody:

1. 3% BSA/TBST (in fridge)

2. Vortex it and then place it in fridge
3. BRCA1- antirabbit (2o AB SOLN) 1 L Ab: 5000 L 3% BSA/TBST Soln
GAPDH- antimouse (2o AB SOLN) 0.5 L Ab: 5000 L 3% BSA/TBST Soln
4. Remove tbst from blots
5. Add ab solutions
6. Incubate 1hour at room temp on rocker (30 RPM)
7. Pour out ab solutions
8. Add TBST, wash 4X for 5 minutes (80-90 RPM)

Secondary ab substrates:

BRCA1 fridge (1ml brown + 1ml clear)

GAPDH Helens Desk (0.5 ml brown + 0.5 ml clear)

1. Turn off lights

2. Pour the clear contents into conical tubes first then brown material (Use clear electronic
pipette, not traditional ones)
3. Take out blots out of wells and place them on cling film
4. Pipette mix onto blot and cover the whole blot
5. Leave it under dark light for 5 minutes

Collect: keys to dark room, scissor, sharpie, timer, films, cassette, blots, DONT BRING PHONE

1. Take blots off cling film, dry them, and add them to cassette, remove as many bubbles as
possible
2. Clean cassette film with ethanol when finished

In dark room:

1. Turn on water, turn off lights

2. Press sheets onto blots, do this for diff int
3. Run through machine

Open image lab:

1. New protocol
2. Select custom film 1 position gel
3. Filter 1 ok
4. Run protocol black and white