PRODUCT-SPECIFIC PROTOCOLS
| WB (UBXN1) | |
|---|---|
| Sample type: | human A431 whole cell, human Jurkat whole cell, human K562 whole cell, human SH-SY5Y whole cell, rat NRK whole cell, rat RH-35 whole cell, mouse NIH/3T3 whole cell, mouse HEPA1-6 whole cell, human Hela whole cell, human HepG2 whole cell, human 293T whole cell, human Ramos whole cell, rat liver tissue, rat brain tissue, mouse liver tissue, mouse brain tissue, Hela whole cell |
| Amount of protein loaded: | 30ug |
| Membrane: | NC |
| Transfer type: | Wet |
| Blocking buffer: | 5% Non-fat Milk/ TBS |
| Primary antibody concentration: | 0.25-0.5 μg/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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