Application
Western blot
Sample
Mouse Cell lysate - whole cell (Mature (8weeks) and immature BMMCs)
Gel Running Conditions
Reduced Denaturing (Precast Gel 4-12%, Criterion TGX (Biorad))
Loading amount
2 µg
Treatment
Cells were mixed in Rippa buffer and denaturated in Laemmli buffer 5min à 95°C
Specification
Mature (8weeks) and immature BMMCs
Blocking step
Milk as blocking agent for 1 minute(s) · Concentration: 0.5% · Temperature: 20°C
Other product details
Incubation time
30 minute(s) · Temperature: 20°C · Diluent: TBST-milk 0.5%
Dilution
1/500
Secondary antibody
Dilution
1/2000
Name
Non-Abcam antibody was used: Jackson Immunoresearch, anti-mouse
Host species: Goat
Clonality: Polyclonal
Conjugation: Cy5 ®
Host species: Goat
Clonality: Polyclonal
Conjugation: Cy5 ®
Detection
Detection method
Fluo
Negative control
Sample 1 and 2: Immature BMMCs (1 and 3 weeks in culture)
Exposure
25 minute(s) and 0 second(s)
Bands
Specific: 35 kDa
Positive control
Sample 3: Mature BMMCs (bone marrow derived-cultured mast cells), obtained after 8 week in culture
Additional data
Additional Notes
For protein detection, a Millipore SNAP i.d.™ 2.0 Protein Detection System was used. Blocking step was realized in 20sec by Actively-driven 30ml of TBST-milk 0.5%-goat serum 1%, through the membrane then anti-Tryptase antibody was added (1/500, 30min, RT) in TBST-milk 0.5%. Membrane was washed 3 times (20sec) with 30ml of TBST. Anti-Mouse-CY5 (Fab) was added (1/2000, 10min, RT) in TBST-milk 0.5 then the membrane was washed again 3 times (20sec) with 30ml of TBST.
Detection was completed with an ImageQuant LAS4000 (GE)
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.
Mr. Francois Daubeuf
Verified customer
投稿 Apr 22 2015