PRODUCT-SPECIFIC PROTOCOLS
| WB (FLOT1) | |
|---|---|
| Sample type: | human A431 whole cell lysates, human U251 whole cell lysates, human PC-3 whole cell lysates, human Hacat whole cell lysates, rat brain tissue lysates, rat skin tissue lysates, mouse brain tissue lysates, mouse skin tissue lysates |
| 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
1
Protein Extraction
Cell culture
- 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.
Tissues
- 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.
2
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.
3
Loading and running the gel
- Select a SDS-PAGE gel. Gel percentage is dependent on the size of the protein:
| 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.
4
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).
5
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.
6
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.
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