Product Review: NOVA2 Antibody (AAS99403E) in human neural cancer cell line using Western Blot

Product Page for NOVA2 antibody – N-terminal region (AAS99403E)

Researcher: Dr. Monica Faronato, University of Liverpool
Application: Western blotting
Species+tissue/cell type: Human neural cell line known to express NOVA2
How many ug’s of tissue/cell lysate run on the gel:  10 ug human neural cell lysate
Primary antibody dilution: 1:1000
Secondary antibody: Goat anti-rabbit IRDye 800
Secondary antibody dilution: 1:15,000

Questionnaire:

How do Antibody Verify’s reagents play a role in your experimental goals?

Determining relative expression across cell lines .

How would you rate this antibody on a scale from 1-5 (5=best) and why?

5.  Very good clean signal by immunoblotting.

Would you use this antibody in future experiments?

Yes.

Have you used another antibody which has worked in your application?

No.

Do you believe the information about the reagent on Antibody Verify’s website is correct?

Yes.

If the antibody works, do you plan to use it in future experiments or to publish your data? Why or why not?

Yes.

Sample Description (please include 1) species type, and 2) cell/tissue type, and 3) how much protein loaded for each lane of your gel):

Human. different types of normal and cancer cells. 10ug.

How many different experimental trials were conducted using the antibody sample?

3

What type of experimental sample are you using and how did you preparing it?

Whole cell lysate prepared in Laemmli buffer (2% SDS, 50mM Tris pH 6.8,10% glycerol).

Primary used and dilution:

1 in 1000, overnight incubation at 4°C.

Secondary used and dilution:

Goat anti-rabbit IRDye 800, 1 in 15,000, 1hr room temperature.

Experimental Procedure/Protocols:

‘Cell lysates were prepared by fractionation or by whole cell lysis:

Whole cell lysis: samples were lysed directly on heat block at 110 degrees in Laemmli buffer (2% SDS, 50mM Tris pH 6.8,10% glycerol)

The following amounts of extracts were used for immunoblotting: 10µg whole cell extract.

(*) Blocking buffer: 5% non-fat dry milk in 1X PBS, pH=7.4

(**) Antibody dilution buffer: 5% non-fat dry milk 0.05% PBST

(***) Membrane washing buffer 0.1% PBST

Membranes were blocked in blocking buffer (*) for 1 hour at room temperature.

Membrane pre-treatment: one wash in distilled water for 20 minutes.

The primary antibodies were diluted 1:500 in antibody dilution buffer (**) and incubated overnight with the membranes (in a cold room at 4°C with agitation to ensure homogenous coverage).

The following day, the primary antibody was poured off and the membranes washed 6 times for 5 minutes each time in membrane washing buffer (***).

Secondary antibody (IRDye 800 conjugated) was diluted in (**) and incubated for 1 hour at room temperature.

Secondary antibody was poured off and the membrane washed 6 times for 5 minutes each time in (***).

A Licor Odyssey system was used to image immunoblots.’

What controls were used in your experiment? Please include your positive control:

Cells known to express or not express NOVA2.

How did you store the antibody after re-suspension?

At -20°C in small working aliquots.

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