TITLE: Western Blot Protocol
AUTHORS: Victoria Beja-Glasser

[STEPS]
?. [Protein lysis]
Made lysis buffer.    o    1:100 DF PPI enzyme in RIPA buffer
?. [Protein lysis]
Placed brains on metal tube holder, which is on ice, and added 500µL lysis buffer. Transferred brain and lysis buffer to thehomogenizer, then homogenized with equipment on Minqing's/Tobias's bench.
?. [Protein lysis]
Transferred homogenate back to the original tube. Then, added 500 uL lysis buffer to clean the glass tube and placed that 500µL to the tube with the brain homogenate (1,000µL total). Mixed homogenate by inverting the tubes.
?. [Protein lysis]
Sonicated 3 cycles of 10 pulses while samples are on ice. Between each cycle, mix tube by inverting.
?. [Protein lysis]
Used these samples for the ThermoFisher BCA assay protocol at 1:5 DF, 1:10 DF, and 1:20 DF.
?. [Thermo BCA assay]
Prepare standards according to the table below using BSA from one ampule in the kit. The diluent should be the same lysis buffer used to prepare protein samples.  Tube    Diluent Volume (µL)    Volume/Source of BSA (µL)    Final [BSA] (µg/mL)    1    0    300 from stock BSA    2000    2    125    375 from stock BSA    1500    3    325    325 from stock BSA    1000    4    175    175 from tube 2    750    5    325    325 from tube 3    500    6    325    325 from tube 5    250    7    325    325 from tube 6    125    8    400    100 from tube 7    25    9    400    0    0   NOTE: I typically will load these standards in the plate in reverse order.
Tube    Diluent Volume (µL)    Volume/Source of BSA (µL)    Final [BSA] (µg/mL)    1    0    300 from stock BSA    2000    2    125    375 from stock BSA    1500    3    325    325 from stock BSA    1000    4    175    175 from tube 2    750    5    325    325 from tube 3    500    6    325    325 from tube 5    250    7    325    325 from tube 6    125    8    400    100 from tube 7    25    9    400    0    0
?. [Thermo BCA assay]
Calculate reagent A and reagent B volumes according to equations below.     Reagent A volume = (# of standards + # of samples) x (# of replicates) x (200µL)     Reagent B volume = (Reagent A volume)/50
?. [Thermo BCA assay]
Mix reagent A with reagent B and vortex to make BCA working reagent.
?. [Thermo BCA assay]
Add 2.5 µL sample + 22.5 µL lysis buffer or 25 µL standard to individual wells of a 96 well flat-bottom plate.
?. [Thermo BCA assay]
Add 200 µL BCA working reagent to each well.
?. [Thermo BCA assay]
Place lid on plate and mix for 30 seconds using plate reader.
?. [Thermo BCA assay]
Incubate at 37C for 30 minutes.
?. [Thermo BCA assay]
Read absorbances using plate reader.
?. [Thermo BCA assay]
Calculate concentrations using BCA Assay spreadsheet.
?. [Western blot (day 1)]
Boil proteins for 10 minutes at 95°C, 65°C, or RT.Cool to room temperature.    o    The boiling temperature is dependent on the type of protein.
?. [Western blot (day 1)]
Spin down samples in microcentrifuge.
?. [Western blot (day 1)]
Assemble vertical gel apparatus.    Remove gel from package and rinse with distilled water.    Remove green strip from bottom of gel.    Gently remove comb. Straighten wells, if necessary.
?. [Western blot (day 1)]
Pour running buffer over wells of gel to rinse out storage buffer. Make sure internal chamber is completely full.    o    Running buffer = 400mL dH20 + 100mL 5X Running Buffer
?. [Western blot (day 1)]
Add 5µL Precision Plus Protein Kaleidoscope Prestained Protein Standard to appropriate wells.    NOTE: If using a small number of samples, avoid edge wells and load protein standard on both sides of experimental samples.
?. [Western blot (day 1)]
Load gel slowly using gel-loading tips.
?. [Western blot (day 1)]
Run gel at 100V for 10 minutes.
?. [Western blot (day 1)]
Increase voltage to 200V and run until dye front runs off of gel. (~20-30 min)
?. [Western blot (day 1)]
Prepare blotting items. Rinse pads in dH2O. Equilibrate all transfer components in transfer buffer. Equilibrate nitrocellulose membrane in sterile dH2O first and then transfer buffer.    o    Transfer buffer = 200mL Methanol + 200mL 5X Transfer buffer + 600mL dH20
?. [Western blot (day 1)]
Place clear side of transfer apparatus in reservoir of transfer buffer.
?. [Western blot (day 1)]
Stack transfer items in the following order in transfer buffer:    i.    Clear side    ii.    1 sponge pad    iii.    2 pieces of filter paper    iv.    Nitrocellulose (notch at top right)    v.    Gel (lane 1 on left)    vi.    2 pieces of filter paper            1.    Roll in all directions to remove bubbles    vii.    Sponge pad    viii.    Black side
?. [Western blot (day 1)]
Clamp shut and place in unit with black to black.
?. [Western blot (day 1)]
Place transfer apparatus in tank.
?. [Western blot (day 1)]
Add ice pack to the tank and fill to thevery top with transfer buffer.
?. [Western blot (day 1)]
Place tank in secondary container and surround with ice.
?. [Western blot (day 1)]
Running conditions…    i.    For proteins     ii.    For proteins >200kDa, transfer at 20V overnight in the cold room.        ** Minqing's suggestion: run the transfer for 2h at 110V.
?. [Western blot (day 1)]
Disassemble transfer apparatus and trim/cut membrane.
?. [Western blot (day 1)]
Place membrane in blocking buffer for 1 hour at RT on orbital shaker.     o    Block buffer = 5% dry milk in TBST    o    TBST = 500µL 10% Tween-20 + 100mL 10X TBS + fill up to 1,000mL dH20
?. [Western blot (day 1)]
Aspirate or pour off blocking buffer.
?. [Western blot (day 1)]
Incubate overnight in blocking buffer + primary antibody at appropriated dilution at 4°C (cold room) on an orbital shaker.
?. [Western blot Continued (day 2)]
Aspirate and save antibody solution.     i.    Can reuse antibody 3-5 times but must store in final concentration 0.02% sodium azide to prevent fungal contamination.    ii.    Menglong also told me that you can freeze the antibody blots at -20C. However, do no use it more than 5 times.
?. [Western blot Continued (day 2)]
Quickly wash 3 times with 1X TBST, pouring off immediately.
?. [Western blot Continued (day 2)]
Wash 3 times for 15 minutes each at RT with 1X TBST on orbital shaker.
?. [Western blot Continued (day 2)]
Add appropriate secondary Ab diluted in blocking buffer and incubate at RT while rocking for 1-2 hours.     a.    Use the buffer made by the company (Odyssey TBS-based blocking buffer, which is located in the deli fridge by Tobias’s/Minqing’s lab bench)    b.    This and all remaining steps should be done in the dark to avoid photobleaching.
?. [Western blot Continued (day 2)]
Quickly wash 3 times with 1X TBST, pouring off immediately.
?. [Western blot Continued (day 2)]
Wash 3 times for 15 minutes at RT with 1X TBST while shaking.
?. [Western blot Continued (day 2)]
Image membrane with Odyssey CLx Infrared Imager.