Q: For Western blot protocol for ABCA1 antibody NB400-105, why does it suggest treating with beta-ME while not heating?

A: For some membrane proteins it is not recommended to boil the samples. You may incubate them at room temperature or at 37C in SDS sample buffer with BME. However, from personal experience, if you boil the samples you will not see the protein on the blot. I do not think the exact reason is known.

Q: Our customer would like to consult something about the ABCA1 antibody with the Cat No. NB400-105. In the datasheet, it showed this protein expressed very low in most cell types. The customer may test the mouse liver tissues, and he doesn’t know the expression level of ABCA1 in this tissue. Is it necessary to induce the expression via LXR treatment to get enough protein expressed? If he doesn’t induce the expression, how much protein loaded was suitable as recommended?

A: Our own Western blot data for NB400-105 was generated by treating RAW cells with 9-cis-retinoic acid and 22R-hydroxycholesterol, which are both known to induce expression of ABCA1 in macrophages. 40ug of protein was loaded on to the gel in this instance. Unfortunately we do not have any testing data from mouse liver tissues using this particular antibody, however the data for another of our ABCA1 antibodies may be of interest to your customer: NB100-2068. The Western blot data for this product was generated using mouse liver tissue, and a clear band can be seen at the expected molecular weight in wild type tissue. We loaded 30ug of protein when running this Western blot, however, your customer may need to optimise the staining procedure for their sample type.

Q: To do Western blot for the determination of ABCA1 protein level, do I need to order NB400-105 or NB400-105B? Which one works better? Or which one you recommend if we also order the secondary antibody catalog # NB730-H?

A: The difference between these two products is biotin conjugation. It is up to you whether you would like to order the primary antibody that is conjugated or not. With the biotin-conjugated antibody (catalog # NB400-105B), you won't need a secondary, just a (strept)avidin-labelled detection reagent, such as streptavidin-HRP. With the unconjugated primary (catalog # NB400-105), you will need to use a secondary antibody. Some people say that when you use the secondary, there is an amplification of signal, because more than one secondary can bind to each primary. However, with the directly conjugated antibody you have fewer reagents and fewer wash steps.

ABCA1 Antibody [Biotin]

Novus Biologicals | Catalog # NB400-105B

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Product Specifications
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Flow Cytometry
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Background
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Research Areas

Key Product Details

Species Reactivity

Human, Mouse, Rat, Porcine, Canine, Chicken, Chinese Hamster, Equine, Hamster, Mustelid, Primate, Rabbit

Applications

Knockout Validated, Immunohistochemistry, Immunohistochemistry-Paraffin, Immunohistochemistry-Frozen, Western Blot, Immunoblotting, ELISA, Block/Neutralize, Flow Cytometry, Dual RNAscope ISH-IHC, Immunocytochemistry/ Immunofluorescence, Immunoprecipitation, Chromatin Immunoprecipitation, Chromatin Immunoprecipitation (ChIP), Gel Super Shift Assays, Gel Supershift Assay, Knockdown Validated, PCR

Label

Biotin

Antibody Source

Polyclonal Rabbit IgG

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Product Specifications

Immunogen

Partial peptide sequence (within residues 1100-1300) of human ABCA1 Antibody [UniProt# O95477]. Actual immunogen sequence is proprietary information.

Reactivity Notes

Canine reactivity reported in scientific literature (PMID: 24612239). Rabbit reactivity reported in scientific literature (PMID: 26444796). Chinese Hamster reactivity reported in scientific literature (PMID: 27902765). Primate reactivity reported in scientific literature (PMID: 25440061). Equine reactivity reported in scientific literature (PMID: 26711702).

Localization

Astrocyte Marker

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG