Since ABCB6 is known to be localized to the mitochondria, what specific considerations should be made for sample preparation (e.g., cell lysis) when using this antibody for Western blotting or immunoprecipitation?

When targeting mitochondrial proteins like ABCB6, it's crucial to ensure efficient lysis of mitochondria to release the protein. For Western blotting, robust lysis buffers containing detergents that effectively solubilize mitochondrial membranes (e.g., RIPA buffer) are generally recommended. For immunoprecipitation, a milder lysis buffer might be preferred to preserve protein complexes, but ensure it still adequately solubilizes the mitochondrial membrane to make the epitope accessible. Mechanical disruption (e.g., Dounce homogenization) can also be used in conjunction with chemical lysis to ensure complete breakdown of mitochondrial structures.

I am experiencing weak or no signal in my immunofluorescence experiments with the Anti-ABCB6 antibody. What troubleshooting steps would you recommend?

Weak or no signal can result from several factors. First, confirm that your cells express ABCB6. Second, ensure proper permeabilization of your cells to allow the antibody access to the mitochondrial antigen. Common permeabilization agents include Triton X-100 or saponin. Third, optimize your primary and secondary antibody concentrations by titration. Fourth, ensure your fixation method is appropriate; over-fixation can mask epitopes. Finally, check the integrity of your secondary antibody and the sensitivity of your detection system. Extending primary antibody incubation time (e.g., overnight at 4℃) can also enhance signal.

Mouse Anti-ABCB6 Recombinant Antibody (V2-178983) (CBMAB-A0218-YC)

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Datasheet

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Datasheet Target References Q & As Review & reward Protocols Associated Products

Basic Information

Host Animal

Mouse

Clone

V2-178983

Application

ELISA, WB

Immunogen

ABCC6 (NP_001162, 831-930 aa) partial recombinant protein with GST tag.

Specificity

Human

Antibody Isotype

IgG1, κ

Clonality

Monoclonal

Application Notes

The COA includes recommended starting dilutions, optimal dilutions should be determined by the end user.

Formulations & Storage [For reference only, actual COA shall prevail!]

Format

PBS, pH 7.2

Buffer

PBS, pH7.2

Preservative

None

Concentration

Batch dependent

Storage

Store at 4°C short term (1-2 weeks). Aliquot and store at -20°C long term. Avoid repeated freeze/thaw cycles.

More Infomation

Target

Full Name

ATP-binding cassette, sub-family B (MDR/TAP), member 6

Introduction

ABCB6 is a member of the ATP-binding cassette (ABC) transporter superfamily. ABC proteins transport various molecules across extra- and intra-cellular membranes. This protein is a member of the heavy metal importer subfamily and plays a role in porphyrin

Entrez Gene ID

10058

UniProt ID

Q9NP58

Alternative Names

ATP Binding Cassette Subfamily B Member 6 (Langereis Blood Group); ATP-Binding Cassette, Sub-Family B (MDR/TAP), Member 6 (Langereis Blood Group); Ubiquitously-Expressed Mammalian ABC Half Transporter; ATP-Binding Cassette Half-Transporter; Mitochondrial

Function

Binds heme and porphyrins and functions in their ATP-dependent uptake into the mitochondria. Plays a crucial role in heme synthesis.

Biological Process

Brain development
Cellular iron ion homeostasis
Heme transport
Porphyrin-containing compound biosynthetic process
Skin development
Transmembrane transport

Cellular Location

Golgi apparatus membrane; Endosome membrane; Cell membrane; Mitochondrion outer membrane; Endoplasmic reticulum membrane. Localized to the endosome-like compartment and dendrite tips.

Involvement in disease

A disorder of eye formation, ranging from small size of a single eye to complete bilateral absence of ocular tissues. Ocular abnormalities like opacities of the cornea and lens, scaring of the retina and choroid, and other abnormalities may also be present. Ocular colobomas are a set of malformations resulting from abnormal morphogenesis of the optic cup and stalk, and the fusion of the fetal fissure (optic fissure).
An autosomal dominant pigmentary genodermatosis characterized by a mixture of hyperpigmented and hypopigmented macules distributed randomly over the body, that appear in infancy or early childhood. The trunk and extremities are the dominant sites of abnormal pigmentation. Facial lesions can be seen in 50% of affected individuals, but involvement of palms and soles is unusual. Abnormalities of hair and nails have also been reported. Dyschromatosis universalis hereditaria may be associated with abnormalities of dermal connective tissue, nerve tissue, or other systemic complications.
A dominantly inherited condition characterized by increased serum potassium levels, measured in whole-blood specimens stored at or below room temperature. This condition is not accompanied by clinical symptoms or biological signs except for borderline abnormalities of red cell shape.

Wang, C., Cao, C., Wang, N., Wang, X., Wang, X., & Zhang, X. C. (2020). Cryo‐electron microscopy structure of human ABCB6 transporter. Protein Science, 29(12), 2363-2374.

Zhang, J., Zhang, X., Li, J., & Song, Z. (2020). Systematic analysis of the ABC transporter family in hepatocellular carcinoma reveals the importance of ABCB6 in regulating ferroptosis. Life Sciences, 257, 118131.

Mares, L., Vilchis, F., Chávez, B., & Ramos, L. (2019). Corrigendum to “isolation and sex steroid effects on the expression of the ATP-binding cassette transporter ABCB6 in Harderian glands of hamster (Mesocricetus auratus)”[Comparative Biochemistry and Physiology, Part A 232 (2019) 40–46]. Comparative Biochemistry and Physiology, Part A, 232, 40-46.

Tessman, R. T. (2019). The Role of Mitochondrial ATP-Binding Cassette Transporter ABCB6 in Metabolism and Energy Balance (Doctoral dissertation, University of Kansas).

Boswell-Casteel, R. C., Fukuda, Y., & Schuetz, J. D. (2018). ABCB6, an ABC transporter impacting drug response and disease. The AAPS journal, 20(1), 1-10.

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Please try the standard protocols which include: protocols, troubleshooting and guide.

Enzyme-linked Immunosorbent Assay (ELISA)
Flow Cytometry
Immunofluorescence (IF)
Immunohistochemistry (IHC)
Immunoprecipitation (IP)
Western Blot (WB)
Enzyme-Linked Immunospot (ELISpot)
Proteogenomics
Other Protocols

Isotype control

For research use only. Not intended for any clinical use.

Custom Antibody Labeling

We also offer labeled antibodies developed using our catalog antibody products and nonfluorescent conjugates (HRP, AP, Biotin, etc.) or fluorescent conjugates (Alexa Fluor, FITC, TRITC, Rhodamine, Texas Red, R-PE, APC, Qdot Probes, Pacific Dyes, etc.).

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