Do you have a protocol for Flow cytometry analysis with this ABCA1 antibody (NB400-105)?

Please see this link to our protocol for Flow cytometry. This is a general protocol that our lab uses as a guideline.

We purchased this antibody a few weeks ago, and we by mistake stored it at -20 degrees Celsius. Is there possible the antibody still works?

Most likely the antibody should still work, but we cannot guarantee it. The biggest thing with antibody storage in most cases, especially for unconjugated antibodies, will be to avoid freeze/thaw cycles as much as possible. So even with ones that we recommend to freeze long term we still recommend aliquoting so they can go through as few of those as possible. I would say since it will probably only undergo one when you move it back to 4 degrees Celsius you should be fine, but again I can't say with 100% certainty. If you have a problem with detection using this product, please let us know so that we can figure out other possibilities to help you in that situation.

I am interested in looking at this marker on monocytes by flow cytometry. I noticed that a directly conjugated 488 ab is available, and that your flow cytometry protocol calls for methanol fixation of the cells. Is the fixation required for antibody binding, or may it be used on cells that have not been methanol fixed? I would like to retain other markers.

Methanol is not required for antibody binding, but it is a fixation method that we found to not interfere with the antibody-antigen binding. You may use unfixed cells, but you may not be able to achieve staining. If you fix your cells with a formaldehyde-based fixative (we recommend antigen retrieval for IHC). However, as an inter-membrane protein, you will need to permeabilize your cells in order for the antibody to access the protein.

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

The difference between NB400-105 or NB400-105B 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.

We purchased NB400-105, Lot# Z5, but the datasheet (approx. 450kDa) was differ from your website (approx. 220kDa) for size of WB. Which molecular weight is correct?

ABCA1 is highly glycosylated and has a predicted molecular weight of approximately 250 kDa without post-translational modification. The apparent molecular weight will change based on treatment and sample. All of the images on our website and datasheet are correct.

Does this product contain any BSA?

No, this antibody does not have any BSA in its formulation.

I bought ABCA1 antibody (NB400-105) from your company recently and used it in Flow cytometry. I want to know the binding site of ABCA1 and the antibody; is it within the cell or outside? Also, do I need to change the cytomembrane's permeabilization before my test?

The immuogen region of NB400-105 is within residues 1100-1300 of human ABCA1 and as a result it does not cover the extracellular domains, as a consequence, permeabilization is required for this antibody. As a general guideline, we usually recommend using 0.5-1.0% Triton detergent in the buffers in order to allow full permeabilization of our antibody.

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?

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.

Is post-nuclear lysate is necessary? Can't I lysate my cells with RIPAbuffer (#R0278 sigma)?

The post-nuclear lysate you mentioned is posted on our website to demonstrate how we do the experiments, and how it was done by our collaborator (Ref Link #1). The procedure of getting the post-nuclear lysate was just for removing the nuclei before lysis and therefore the cleaner the lysates would be. You do not need to follow the method for the experiment that led to the result.

Why shouldn't I boil the samples? is it OK to boil them if using RIPAbuffer?

To fractionate a membrane-bound protein, the lipid moieties associated with the protein frequently cause the smearing or streaky appearance of our WB. To result this difficulty, it is advised that we resuspend cell pellets in the SDS-sample buffer, left at room temp without boiling, spinning down the viscous cell debris, and then subject to PAGE and blotting. And this was the reason we suggested you not to boil your protein samples. Whether it will work for resuspending cell pellets in RIPA buffer, unfortunately we have no such data.

Can i use DTT instead of Beta- mercaptoethanol?

Either DTT or 2-mercaptoethanol is routinely used in SDS-PAGE sample buffer (reducing gel electrophoresis). Except that 2-mercaptoethanol is a stronger reducing agent and is mostly frequently used for that purpose.

Have you tested abca1 antibody(catalog # NB400-105) on hepatic Abca1 expression?

We do not currently have any testing data where we have used NB400-105 with hepatic samples; all of our testing data for NB400-105 can be seen on the product page for this antibody. Should your customer be interested, we do show murine liver data with the following product: NB100-2068.

ABCA1 Antibody - BSA Free

Name: ABCA1 Antibody - BSA Free
Brand: Novus Biologicals
SKU: NB400-105
Rating: 4 (7 reviews)

Novus Biologicals, part of Bio-Techne | Catalog # NB400-105

(7)

Citations (415)

Product Datasheet / COA / SDS / Protocols & Manuals

View Available Conjugates

Conjugate

Catalog #

Alexa Fluor 350

NB400-105AF350

View Product

Alexa Fluor 405

NB400-105AF405

View Product

Alexa Fluor 488

NB400-105AF488

View Product

Alexa Fluor 532

NB400-105AF532

View Product

Alexa Fluor 594

NB400-105AF594

View Product

Alexa Fluor 647

NB400-105AF647

View Product

Alexa Fluor 700

NB400-105AF700

View Product

Alexa Fluor 750

NB400-105AF750

View Product

Allophycocyanin

NB400-105APC

View Product

Allophycocyanin/Cy7

NB400-105APCCY7

View Product

Biotin

NB400-105B

View Product

DyLight 350

NB400-105UV

View Product

DyLight 405

NB400-105V

View Product

DyLight 488

NB400-105G

View Product

DyLight 550

NB400-105R

View Product

DyLight 594

NB400-105DL594

View Product

DyLight 650

NB400-105C

View Product

DyLight 680

NB400-105FR

View Product

DyLight 755

NB400-105IR

View Product

FITC

NB400-105F

View Product

HRP

NB400-105H

View Product

Janelia Fluor 549

NB400-105JF549

View Product

Janelia Fluor 646

NB400-105JF646

View Product

mFluor Violet 450 SE

NB400-105MFV450

View Product

mFluor Violet 500 SE

NB400-105MFV500

View Product

mFluor Violet 610 SE

NB400-105MFV610

View Product

NB400-105PE

View Product

PE/Atto594

NB400-105PEATT594

View Product

PE/Cy5.5

NB400-105PECY55

View Product

PE/Cy7

NB400-105PECY7

View Product

PerCP

NB400-105PCP

View Product

100% Guarantee

Product Specifications
Scientific Data
Applications
Preparation & Storage
Background
Product Documents
Specific Notices

Key Product Details

Validated by

Knockout/Knockdown, Independent Antibodies, Tagged Protein Expression

Species Reactivity

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

Applications

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

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Format

BSA Free

Concentration

1 mg/ml

View all ABCA1 Antibodies »

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

Theoretical MW

220 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Scientific Data Images for ABCA1 Antibody - BSA Free

Dual RNAscope ISH-IHC: ABCA1 Antibody [NB400-105] - Formalin-fixed paraffin-embedded tissue sections of human prostate cancer were probed for ABCA1 mRNA (ACD RNAScope Probe, catalog # 432291; Fast Red chromogen, ACD catalog # 322360). Adjacent tissue section was processed for immunohistochemistry using rabbit polyclonal (Novus Biologicals catalog # NB400-105) at 1.5ug/mL with overnight incubation at 4 degrees Celsius followed by incubation with anti-rabbit IgG VisUCyte HRP Polymer Antibody (Catalog # VC003) and DAB chromogen (yellow-brown). Tissue was counterstained with hematoxylin (blue). Specific staining was localized to glandular cells.

Knockdown Validated: ABCA1 Antibody [NB400-105] - HSKMCs were transfected with scrambled siRNA (scr siRNA) or ABCA1 siRNA. The cells were lysed and ABCA1 (A) protein levels were quantified by immunoblotting. Image collected and cropped by CiteAb from the following publication (http://www.nature.com/articles/s41598-018-38014-3) licensed under under a CC-BY license.

Western Blot: ABCA1 Antibody [NB400-105] - Analysis of ABCA1 in total cell lysates of RAW264.9 cells treated with vehicle (-) or 9-cisretinoic acid and 22Rhydroxycholesterol (+). Samples used for this testing were 40 ug of total cell post-nuclear lysate from each group.

Immunocytochemistry/Immunofluorescence: ABCA1 Antibody [NB400-105] - HepG2 cells were grown to 60% confluency, serum starved for 24 hours, and then treated with 1uM TO9 for 24 hours prior to being fixed for 10 minutes using 10% formalin and then permeabilized for 5 minutes using 1X TBS + 0.5% Triton-X100. The cells were incubated with anti-ABCA1 at 5.0ug/ml overnight at 4C and detected with an anti-rabbit Dylight 488 (Green) at a 1:500 dilution. Alpha tubulin (DM1A) NB100-690 was used as a co-stain at a 1:1000 dilution and detected with an anti-mouse Dylight 550 (Red) at a 1:500 dilution. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.

Immunocytochemistry/Immunofluorescence: ABCA1 Antibody [NB400-105] - Untreated HepG2 cells were grown to 60% confluency, and serum starved for 24 hours prior to being fixed for 10 minutes using 10% formalin and then permeabilized for 5 minutes using 1X TBS + 0.5% Triton-X100. The cells were incubated with anti-ABCA1 at 5.0ug/ml overnight at 4C and detected with an anti-rabbit Dylight 488 (Green) at a 1:500 dilution. Alpha tubulin (DM1A) NB100-690 was used as a co-stain at a 1:1000 dilution and detected with an anti-mouse Dylight 550 (Red) at a 1:500 dilution. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.

Flow Cytometry: ABCA1 Antibody [NB400-105] - ABCA-1 FITC conjugated antibody of human adipose tissue macrophage subsets by flow cytometry. Image from verified customer review.

Immunohistochemistry-Paraffin: ABCA1 Antibody [NB400-105] - Association between expression of ABCA1 and survival in ovarian cancer patients. Expression of ABCA1 in 55 ovarian cancer patient samples was determined by IHC in tissue microarray. Representative image of ovarian cancer showing high (left panel) and low (right panel) ABCA1 expression on the cell membrane or cytoplasm (x400). Image collected and cropped by CiteAb from the following publication (http://www.clinicalepigeneticsjournal.com/content/7/1/1), licensed under a CC-BY license.

Western Blot: ABCA1 Antibody [NB400-105] - ABCA1 in human primary fibroblasts in culture. 10 ug of total protein. 7.5% TGX gel. Blocking 5% milk in PBST 1h RT. 1:1000 primary ab in BSA 3% PBST O/N at 4C. Secondary 1:5000 HRP 1h RT. Arrow shows around 250 kDa. WB image submitted by a verified customer review.

Flow Cytometry: ABCA1 Antibody [NB400-105] - ABCA1 antibody was tested at 1: 400 in HeLa cells using an Alexa Fluor 488 secondary (shown in purple). M1 is defined by unstained cells.

Western Blot: ABCA1 Antibody [NB400-105] - Detection of ABCA1 in mouse peritoneal macrophages using NB 400-105 (Lot L). ECL exposure, 1 min. Lane 4: T09 uninduced lysate Lane 5: T09 induced lysate.

Western Blot: ABCA1 Antibody [NB400-105] - Prostates of LXR mutant mice accumulate cholesterol esters through inappropriate LXR target genes regulation. Total protein lysates of WT and LXR null mice under normal or high cholesterol diet were analyzed by western blotting with antibodies against ABCA1, LDLR and ACTIN as a loading control (left panel), quantification of ABCA1 and LDLR protein accumulation levels (right panel). * p0.05, *** p0.001 in Student's t test. Error bars represent the +/- mean SEM. Image collected and cropped by CiteAb from the following publication (http://dx.plos.org/10.1371/journal.pgen.1003483), licensed under a CC-BY license.

Western Blot: ABCA1 Antibody [NB400-105] - miR-33b reduces cellular cholesterol efflux and serum HDL-C levels. Western blotting for ABCA1 (using NB400-105) and ABCG1 proteins in peritoneal macrophages from WT and KI+/+ mice. Representative images are shown. beta-actin was used as the loading control. Image collected and cropped by CiteAb from the following publication (http://www.nature.com/articles/srep05312), licensed under a CC-BY license.

Western Blot: ABCA1 Antibody [NB400-105] - miR-33b regulates ABCA1 and SREBP-1. Analysis for ABCA1, SREBP-1, CPT1a, and AMPK alpha protein levels in the livers of WT, KI+/-, and KI+/+ mice. Representative images are shown. TF2B and beta-actin were used as loading controls. Image collected and cropped by CiteAb from the following publication (http://www.nature.com/articles/srep05312), licensed under a CC-BY license.

Immunohistochemistry-Paraffin: ABCA1 Antibody [NB400-105] - Detection of ABCA1 in human prostate epithelium showing luminal and membrane staining.

View 11 more images

Applications for ABCA1 Antibody - BSA Free

Application

Recommended Usage

Block/Neutralize

reported in scientific literature (PMID 30821416)

Chromatin Immunoprecipitation

reported in scientific literature (PMID 19515742)

ELISA

reported in scientific literature (PMID 18541924)

Flow Cytometry

1:400. Use reported in scientific literature (PMID 21501868)

Gel Super Shift Assays

reported in scientific literature (PMID 15684432)

Immunoblotting

reported in scientific literature (PMID 27599291)

Immunocytochemistry/Immunofluorescence

1:100. Use reported in scientific literature (PMID 21501868)

Immunohistochemistry

1:200

Immunohistochemistry-Paraffin

1:200

Immunoprecipitation

1:10-1:500. Use reported in scientific literature (PMID 21846716; 21106520)

Knockdown Validated

reported in scientific literature (PMID 31666189)

Knockout Validated

reported in scientific literature (PMID 32273567)

PCR

reported in scientific literature (PMID 27406916)

Simple Western

1:50

Western Blot

1:500

Application Notes

Western Blot band representing ABCA1 is observed at approx. 220 kDa. Additional non-specific bands are seen at lower molecular weights, but do not interfere with the ABCA1 signal. In Simple Western only 10-15 uL of the recommended dilution is used per data point. Separated by Charge.

Reviewed Applications

Read 7 reviews rated 4.3 using NB400-105 in the following applications:

Immunofluorescence (1 Review)
Western Blot (6 Reviews)

Formulation, Preparation, and Storage

Purification

Immunogen affinity purified

Formulation

PBS

Format

BSA Free

Preservative

0.02% Sodium Azide

Concentration

1 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.

Background: ABCA1

ATP-binding cassette transporter A1 (ABCA1) is a cAMP-dependent and sulfonylurea-sensitive anion transporter belonging to the ATP-binding cassette family. ABCA1 is involved in the regulation of apolipoprotein AI (apoAI)-mediated cholesterol efflux and high-density lipoproteins (HDL) metabolism (1). In the brain, ABCA1 transports cholesterol to apoE, the major CNS apolipoprotein, where it influences motor function and synaptic morphology.

ABCA1 is comprised of 2,261 amino acids with a theoretical molecular weight of 254 kDa and human ABCA1 shares 97% amino acid identity with mouse ABCA1. The general structure of ABCA consists of two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). ABCA1 is a widely distributed cell-membrane protein with the highest expression found in macrophages. DHHC8 mediated palmitoylation of ABCA1 is essential for its localization to the plasma membrane and expression of mouse ABCA1 (not human) is induced by cAMP analogs (2). ABCA1 is phosphorylated at Ser1042 and Ser2054 by PKA, with Ser2054 being key for regulating phospholipid efflux. Mutations in ABCA1 have been linked to atherosclerosis and the progression of metabolic syndrome phenotypes: high density lipoprotein deficiency type 1 (HDLD1); also known as Tangier disease (TGD), and high density lipoprotein deficiency type 2 (HDLD2); also known as familial hypoalphalipoproteinemia (FHA) (3).

References

1.Oram JF, Lawn RM. (2001) ABCA1. The gatekeeper for eliminating excess tissue cholesterol. J Lipid Res. 42(8):1173-9. PMID: 11483617

2.Singaraja RR, Kang MH, Vaid K, Sanders SS, Vilas GL, Arstikaitis P, Coutinho J, Drisdel RC, El-Husseini Ael D, Green WN, Berthiaume L, Hayden MR. (2009) Palmitoylation of ATP-binding cassette transporter A1 is essential for its trafficking and function. Circ Res. 105(2):138-47. PMID: 19556522

3.Attie AD. (2007) ABCA1: at the nexus of cholesterol, HDL and atherosclerosis. Trends in Biochemical Sciences 32(4):172-9. PMID: 17324574

Long Name

ATP-binding Cassette, Sub-family A (ABC1), Member 1

Alternate Names

ABC1, CERP, HDLDT1, TGD

Entrez Gene IDs

19 (Human); 11303 (Mouse); 313210 (Rat)

Gene Symbol

ABCA1

UniProt

O95477 (Human)

Additional ABCA1 Products

Product Documents for ABCA1 Antibody - BSA Free

Product Datasheets

Download Product Datasheets

Protocols & Manuals

Download IHC-P Protocol

Download Western Blot Protocol

COA

SDS

Download SDS

Product Specific Notices for ABCA1 Antibody - BSA Free

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Citations
Reviews

Protocols
FAQs

ABCA1 Antibody - BSA Free

Novus Biologicals, part of Bio-Techne | Catalog # NB400-105

Key Product Details

Validated by

Species Reactivity

Applications

Label

Antibody Source

Format

Concentration

View all ABCA1 Antibodies »

Product Specifications

Immunogen

Reactivity Notes

Localization

Clonality

Host

Isotype

Theoretical MW

Scientific Data Images for ABCA1 Antibody - BSA Free

Applications for ABCA1 Antibody - BSA Free

Block/Neutralize

Chromatin Immunoprecipitation

ELISA

Flow Cytometry

Gel Super Shift Assays

Immunoblotting

Immunocytochemistry/Immunofluorescence

Immunohistochemistry

Immunohistochemistry-Paraffin

Immunoprecipitation

Knockdown Validated

Knockout Validated

PCR

Simple Western

Western Blot

Reviewed Applications

Formulation, Preparation, and Storage

Purification

Formulation

Format

Preservative

Concentration

Shipping

Stability & Storage

Background: ABCA1

Long Name

Alternate Names

Entrez Gene IDs

Gene Symbol

UniProt

Additional ABCA1 Products

Product Documents for ABCA1 Antibody - BSA Free

Product Datasheets

Protocols & Manuals

COA

Certificate of Analysis

SDS

Product Specific Notices for ABCA1 Antibody - BSA Free