Western blot abreview for Anti-Glutaminase antibody

Average
Abreviews
Application
Western blot
Sample
Human Cell lysate - whole cell (lymphocytes)
Loading amount
12.5 µg
Specification
lymphocytes
Treatment
activated for 72h (anti-CD3/CD28)
Gel Running Conditions
Reduced Denaturing (8% PA)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Other product details

Dilution
1/1000
Incubation time
16 hour(s) and 0 minute(s) · Temperature: 4°C

Secondary antibody

Name
Non-Abcam antibody was used: anti-rabbit HRP (Dako)
Host species: Donkey
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Dilution
1/500

Detection

Detection method
Millipore ECL
Exposure
5 second(s)
Bands
Specific: ca. 58 kDa Non-specific: 48 and 50 kDa
Positive control
full-length human GLS1 overexpressed in HEK293 (whole cell lysate 1 ug total protein)

Additional data

Additional Notes
GLS1 in lymphocytes was identified as the smaller band at 58 kDa and of exactly the same size as the overexpressed positive control. It remains unclear which protein corresponds to the much stronger band at ca. 50 kDa (also visible in HEK cells).
If it wasn't for the positive control, one may easily misinterpret the bands by just going for the strongest one. Also, the band may become a problem when using gels that do not separate well at 50-60 kDa or when intending to apply this antibody for immunohistochemsitry or flow cytometry.

NOTE: The positive control was run on the same gel, but lanes between the lymphocyte and HEK samples have been cut out and the remaining two stitched together for illustrating purposes.

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Submitted Nov 22 2010

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