Western blot abreview for Anti-HA tag antibody [HA.C5]

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Abreviews
Application
Western blot
Sample
Human Cell lysate - whole cell (HeLa)
Gel Running Conditions
Reduced Denaturing (10% NuPAGE Gel Bis-Tris)
Loading amount
10 µg
Specification
HeLa
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Other product details

Incubation time
20 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 5% PBS-T
Dilution
1/1000

Secondary antibody

Name
Non-Abcam antibody was used: AffiniPure Donkey anti-Mouse IgG (H+L) (Jackson)
Host species: Mouse
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase

Detection

Detection method
ECL
Negative control
Non HA-expressing cells.
Exposure
3 minute(s) and 0 second(s)
Bands
Specific: 55 kDa
Positive control
HA-expressing stable cell line.

Additional data

Additional Notes
Lysis and running: Cells lysed in 1.5X Lammeli buffer +0.15M DTT syringed 10X with 25g needle and then boiled 100 degrees C for 10 minutes. 10ug of sample run out on 4-12% Bis Tris Nupage gel in 1X MOPS buffer. WB protocol: Western blot was performed by wet transfer of gel onto nitrocellulose membrane 0.22u pore size\n 100V for 2 h 4 degrees. Blocked in 5% milk PBST for 1 h room temp (1X PBS + 0.1% tween 20) incubated 24h at 4 degrees in 5% PBST with primary antibody Rabbit polyclonal to CRISPR-Cas9 ab204448) 1:1000. Washed 3X PBST; IgG anti rabbit HRP 1:5000 (Jackson Immunoscience); Washed 3x PBST; Washed 1X PBS; Developed using ECL prime kit (GE Healthcare).

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Submitted Feb 14 2019

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