Application
Western blot
Sample
Human Cell lysate - whole cell (Myometrial smooth muscle cells)
Loading amount
15 µg
Specification
Myometrial smooth muscle cells
Treatment
1 or 10 nM oxytocin for 1 minute, and 1µM phorbol 12-myristate 13-acetate for 10 minutes
Gel Running Conditions
Reduced Denaturing (13% tris-glycine SDS-PAGE gel)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Other product details
Dilution
1/1000
Incubation time
16 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: TBS + 0.1% Tween-20
Secondary antibody
Name
Non-Abcam antibody was used: Anti-rabbit HRP-linked IgG (H+L)
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Dilution
1/5000
Detection
Detection method
ECL
Exposure
1 minute(s) and 0 second(s)
Bands
Specific: 17 kDa Non-specific: 18 kDa
Additional data
Additional Notes
Adhered cells were rinsed in PBS then lysed directly in boiling SDS-PAGE sample buffer (25mM Tris pH 6.8, 2% SDS, 10% glycerol, 12.5 mM DTT).
Proteins were transferred to standard PVDF membrane (no improvement when using reduced pore size membrane) in a buffer containing 25mM Tris, 200mM glycine, 10% methanol and 0.05% SDS for 1 hour at 250mA.
Lanes on Western:
1. Non-stimulated
2. 1nM oxytocin, 1 minute
3. 10nM oxytocin, 1 minute
4. 1µM phorbol 12-myristate 13-acetate, 10 minutes
Proteins were transferred to standard PVDF membrane (no improvement when using reduced pore size membrane) in a buffer containing 25mM Tris, 200mM glycine, 10% methanol and 0.05% SDS for 1 hour at 250mA.
Lanes on Western:
1. Non-stimulated
2. 1nM oxytocin, 1 minute
3. 10nM oxytocin, 1 minute
4. 1µM phorbol 12-myristate 13-acetate, 10 minutes
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
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投稿 Apr 10 2009