Western blot abreview for Anti-NCKAP1L antibody

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Abreviews
Application
Western blot
Sample
Mouse Cell lysate - whole cell (both hematopoietic and adherent cell lines)
Loading amount
20 µg
Specification
both hematopoietic and adherent cell lines
Gel Running Conditions
Reduced Denaturing (10%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Other product details

Dilution
1/100
Incubation time
16 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: TBST + 0.5% Milk

Secondary antibody

Name
Non-Abcam antibody was used: goat-anti-rabbit-Peroxidase coupled
Host species: Goat
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Dilution
1/1000

Detection

Detection method
Roche Lumi-light ECL
Exposure
20 minute(s) and 0 second(s)
Bands
Specific: ca. 110 kDa Non-specific: ca. 65, ca. 30 kDa
Positive control
Human hematopoietic cell lines, e.g. Jurkat and THP-1 cells and B16 cells transfected with NCKAP1L
NB!! These positive controls did not always display a specific NCKAP1L band!! The band pattern of B16 with or without NCKAP1L transfection was also similar.
Negative control
B16 cells not transfected with NCKAP1L

Additional data

Additional Notes
DISCOUNT CODE: 100%ABR-E7BVB

There are concerns whether this antibody is specific to NCKAP1L, because it also gives a "specific" band around the expected height in non-hematopoietic cells. This unexpected, since NCKAP1L expression is as mentioned in the product sheet: "Expressed only in cells of hematopoietic origin". The suggested positive control, 293T cells, should therefore also not express NCKAP1L and would be more likely to be a negative control. Possibly there is cross reactivity with Nap1, which is expressed in adherent cells.
Abcam response
After looking over your protocol it may help to reduce the concentration of the antibody that you are using. We recommend using a 1 ug/ml concentration which would be a 1/1000 dilution. Additionally, adding more of the blocking agent to the primary antibody for the incubation can also help reduce background.

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Submitted Jul 21 2011

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