Application
Western blot
Sample
Human Cell lysate - whole cell (HEK293)
Gel Running Conditions
Reduced Denaturing
Loading amount
15 µg
Specification
HEK293
Blocking step
Thermo Scientific #EN37543 Starting Block TBST formulation as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C
Other product details
Incubation time
16 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 1:5000
Secondary antibody
Dilution
1/10000
Name
Non-Abcam antibody was used: Jackson ImmunoResearch #111-035-045
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Detection
Detection method
ECL
Negative control
Lysates from untransfected cells and cells transfected with empty vector were used as negative controls.
Exposure
10 second(s)
Bands
Specific: none kDa Non-specific: 52, 56 kDa
Positive control
Cells were transfected with V5-tagged protein or empty vector.
Additional data
Additional Notes
We detected a non-specific doublet at 52 and 56 kDa, which was present in all samples, including the negative controls. We stripped the blot and re-probed with an antibody specific to our target protein (also ~55 kDa) and only detected the proper band in samples transfected with the V5-tagged target protein. Thus, this antibody is cross-reacting with a protein present in HEK293 cell lysates.
Abcam response
This antibody is covered by our 12 month Abpromise guarantee to work in Western blot. A replacement, credit, or refund was offered to this researcher.
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.
Abcam user community
Verified customer
投稿 Apr 22 2015