8. Western Blotting Protocol

Post date: May 26, 2018 4:57:43 PM

I. Sample Preparation

a. Get Cells and Make Lysate

i. Remove plated cells from incubator

ii. Prepare and label an epi per well, keep on ice

iii. Use a cell scraper to remove adherent cells

1. Invert plate and look through to confirm all cells removed

iv. Scrape cells alongwith media from wells using cell scraper and deposit into eppendorf tubes

v. Collect Pellet

1. Spin down in cold room at 1000rpm for 5 minutes

2. Remove media, at this point, cells can be stored at -80C

3. Keep cells and media on ice at all times

vi. Wash cells

1. Add 1ml of PBS 1x chilled to each epi

2. Mix with cell pellet and homogenize

3. Spin down in cold room at 1000rpm for 5 minutes

4. Remove supernatant, as much as possible without touching pellet

vii. Add 1X RIPA lysis buffer solution

Recipe to prepare 1X RIPA lysis buffer (Total volume: 1000 ul)

880 ul MilliQ water

100 ul 10x RIPA buffer [EMDMillipore 10X RIPA buffer Cat.# 20-188]

10 ul Protease inhibitor (100x) [ThermoScientific; Halt protease Inhibitor cocktail Cat.# 87786]

10 ul Phosphatase inhibitor (100x) [Sigma; Phosphatase Inhibitor Cocktail 2 Cat.# P5726]

1. Recipe on sticky note (milliQ H2O, 10x RIPA, 100x phosphatase and protease inhibitors)

a. Mix water with RIPA first

b. Add inhibitors only right before use on cells

2. Add quantity of lysate solution as determined by pellet size

a. EX: 35 µL per Eppendorf tube for harvest of 2 wells from 6-well plate

3. Vigorously mix lysate solution with cell pellet

viii. Incubate on Ice

1. Incubate for 30 minutes

2. Vortex every 10 minutes

ix. Collect protein lysate

1. Spin down lysate solution in cold room at 12500rpm for 20 minutes (protein in supernatant)

2. Remove supernatant and save into new Eppendorf tubes

a. Protein solution can now be stored for later use with freezing at -80C

3. Determine protein concentration with BSA assay

a. Take BSA (1mg/1ml) out of -20C and thaw on ice

b. Use 96 well plate and deposit 0,1,2,4,6,8 µL BSA into test wells (duplicate)

c. Prepare BCA Reagent mixture (A:B = 50:1)

i. Need 200µL BCA reagent mixture / well, calculate amount to make accordingly

ii. Add amount of reagent B only right before using

d. Take 1 µL of protein lysate from each epi and deposit into well, run in duplicate

e. Add BCA mixture 200µL per well

f. Pipette mix each solution to eliminate bubbles

g. Incubate at 37C for 30 minutes protect from light

h. Go to 4N and use micro-plate reader. Read the absorbance at 562 nM.

i. Interpret concentrations and analyze volume needed to add 25-30 µg per well for running gel

4. Prepare epi’s with calculated volumes of protein solution, 10x reducing agent (from 4C fridge) and 4x LDS sample buffer

a. Use “bca calculation template” excel file for calculation guiding

b. Prepare Proteins for Electrophoresis

i. Prepare gels ahead of time (protein gels protocol)

ii. Denature protein solution

1. Use heat block at 92C for 8 minutes (keep epis closed!)

2. Spin down to collect condensation

3. Prepare electrophoresis apparatus

a. Use pen to outline wells for easy loading

b. Use running buffer and ensure it covers entire gel

c. Reuse running buffer only once, remake after

4. Withdraw calculated amount of protein needed to load from epis with gel loading pipette tip and deposit into well, USE A P100!!

5. Use 10-15 µL of ladder (-20C fridge) in first column

6. Ensure proper amount of running buffer and then start

II. Electrophoresis

a. Run assembly at 90V for a few minutes

b. Once protein enters separating gel, use 120-130V for approximately 1.5 hours

c. Stop electrophoresis right before proteins run off the gel

III. Transfer and Treatment

a. Visualize proteins on gels

i. Keep gel oriented in familiar position in reference to ladder

ii. Carefully remove big plate from smaller clear plate

iii. Gel should stick to small plate

b. Prepare membrane and sponges and filters

i. Soak PVDF membrane in 100% methanol for two minutes, remove

ii. Soak membrane in transfer buffer for two minutes

iii. Soak sponges and filters in transfer buffer while preparing membrane

c. Assemble apparatus (white/red close to you, black away)

i. Lay wet sponge on white side, lay filter on top of sponge

ii. Lay membrane on next and carefully add gel right on top

iii. Stack another filter and remove bubbles then a sponge

iv. Close the assembly, slide into rectangular box and match with colors

d. Transfer

i. Set up transfer in cold room

ii. Plug in and run

1. Fast run (1.5h) set to 100V

2. Regular run (2h) set to 80V

3. Overnight run (12h) set to 30V

e. Visualize proteins on membrane to confirm complete transfer

f. Blocking the membrane (preventing non-specific binding)

i. Use razor to cut out unwanted sections of membrane

ii. Wash with 1x TBS TWEEN solution for 5 min at room temperature

iii. Use 5% milk (or BSA) and TBST solution (check for precipitation) and incubate for 1 hour at room temperature with agitation (on shaker)

g. Incubation with primary antibody

i. Use antibody (concentration dependent on antibody)+ 5% milk made in 1X TBST solution

ii. Incubate with membrane overnight at 4C agitation OR incubate at room temperature for 2h

iii. If you made primary antibody from scratch, SAVE IT!!

h. Wash 3x

i. Use 1x TBS TWEEN solution

ii. 10 minutes/wash

i. Incubation with secondary antibody (1:5000 dilution for goat anti-mouse antibody)

i. Use 1µL anti-primary antibody (secondary antibody) in 5mls 5% milk and TBST solution

ii. Incubate at room temperature for 1 hour

iii. If you want to make it more concentrated (1:4000) longer wash times are needed to remove background

j. Wash 3x

i. Use 1x TBS TWEEN solution

ii. 10 minutes/wash

IV. Development

a. Use tweezers to place membrane on glass slide

b. Warm ECL Reagents up to room temperature

c. Use ECL Reagents 1 and 2 in 1:1 (for small membrane use ~total 400 µL, for larger membrane use ~ total 600 µL

d. Incubate on glass slide for 3 minutes

e. Dab off membrane and press completely dry

f. Place in binder, use film paper and timer, go to dark room

g. In dark room, place film paper EXACTLY over membrane (expose for seconds to minutes as determined by band observation

h. Return to lab and label film

V. Loading Control with B-actin (42kDa)

i. Protocol for B-actin can be “rushed” because primary antibody binding is quick and strong

b. Wash membrane for 10 minutes with 1x TBS TWEEN solution

c. Cover membrane with stripping buffer and incubate for 10 minutes at room temperature

d. Wash membrane with 1x TBS TWEEN solution for ten minutes

e. Block membrane with 5% milk made in 1X TBST solution for 1h at room temperature (30 minutes if rushed)

f. Apply primary antibody (1:1000 dilution), incubate overnight in cold room OR incubate at room temperature for 1.5h-2h

g. Wash 3x with 1x TBS TWEEN solution for 10 minutes (5 minutes if rushed)

h. Apply secondary antibody (anti-rabbit) (1:5000 dilution) for 1h room temperature (45 minutes if rushed)

i. See step IIIJ

*TBS TWEEN solution = TBS TWEEN 20