PRODUCT-SPECIFIC PROTOCOLS

WB (VCP)
Sample type:	human Hela whole cell, human SH-SY5Y whole cell, human MCF-7 whole cell, human A431 whole cell, human K562 whole cell, human HepG2 whole cell, monkey COS-7 whole cell, rat brain tissue, rat lung tissue, rat stomach tissue, rat C6 whole cell, mouse brain tissue, mouse lung tissue, mouse RAW2647 whole cell, human Hela whole cell, human SH-SY5Y whole cell, human MCF-7 whole cell, human A431 whole cell, human HepG2 whole cell, human K562 whole cell, human U87 whole cell, rat brain tissue, rat lung tissue, rat kidney tissue, rat C6 whole cell, mouse brain tissue, mouse lung tissue, mouse kidney tissue, mouse RAW2647 whole cell
Amount of protein loaded:	30ug
Membrane:	NC
Transfer type:	Wet
Blocking buffer:	5% Non-fat Milk/ TBS
Primary antibody concentration:	0.1-0.5μg/ml
Incubation time:	overnight at 4°C
Detection Method:	ECL

PROTOCOL

Protein Extraction

Scrape cells or trypsinize cells and wash with PBS.
Centrifuge at 3000 rpm for 3 minutes, remove PBS. The centrifugation force and time can be varied depending on the cell type.
Add RIPA lysis buffer, incubate on ice for 30-60 minutes.
Sonicate for 10-15 seconds to complete cell lysis.
Centrifuge at 10000 rpm for 10 minutes. The centrifugation force and time can be varied depending on the cell type.
Aspirate supernatant avoiding pellet and upper, lipid-containing phase. Aliquot supernatant and store at -20°C.

Mince tissue and then rinse tissue in ice-cold PBS.
Place minced tissue in tissue homogenizer.
Add ice-cold RIPA lysis buffer. For a 0.5g piece of tissue, add 5ml of ice-cold lysis buffer (volumes of lysis buffer must be determined in relation to the amount of tissue present). Incubate on ice for 30-60 minutes.
Homogenize for several minutes at high speed.
Sonicate until no tissue chunks remain.
Centrifuge at 10000 rpm for 10 minutes. The centrifugation force and time can be varied depending on the sample type.
Aspirate supernatant.
Aliquot supernatant and store at -20°C.

Sample preparation

Remove the quantity required for use.
Determine how much protein to load and add an equal volume of 2X SDS-PAGE Loading Buffer.
Heat the sample to 100°C for 5 minutes.

Loading and running the gel

Protein Size (kDa)	>100	30-100	10-30	<10
Gel Percentage	8%	10%	12%	15%

Add an appropriate amount of SDS-PAGE Electrophoresis Buffer into the electrophoresis chamber.
Load equal amounts (30 ug) of protein into the wells of the SDS-PAGE gel, along with appropriate molecular weight marker.
Electrophorese proteins for 2-3 hours at an appropriate voltage.

Transferring the protein from the gel to the membrane

Place the gel in transfer buffer for 5-10 minutes.
Prepare membrane (NC or PVDF). Wet PVDF membrane with methanol for 15 seconds and place membrane in transfer buffer for 5 minutes.
Apply a constant current of 150mA for an appropriate time, 40-120 min (transfer time must be optimized according to gel concentration).

Blocking

Wash the membrane in three washes of wash buffer, 10 minutes each.
Block the membrane on shaker for 1.5 hours at room temperature using 5% Non-fat Milk/TBS.

Incubation with primary antibody and secondary antibody

Dilute primary antibody with antibody diluent to the concentration recommended.
Incubate the membrane with primary antibody overnight at 4°C.
Wash the membrane with TBS-0.1%Tween 3 times, 5 minutes each.
Prepare conjugated secondary antibody at pre-determined dilution in diluent buffer.
Incubate the membrane with goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hours at RT.
Wash the membrane in three washes of wash buffer, 10 minutes each.