Given that ABCB4 is a multi-pass transmembrane protein, what are the best practices for sample preparation for Western blotting to ensure optimal detection?

For optimal detection of ABCB4, adequate solubilization of the membrane protein is crucial. We recommend using robust lysis buffers containing strong detergents (e.g., RIPA buffer with SDS) to ensure complete cell lysis and protein solubilization. For tissues, mechanical homogenization followed by detergent lysis is often necessary. Ensure your samples are heated appropriately (e.g., 95-100℃ for 5-10 minutes) in SDS-PAGE loading buffer to fully denature and reduce the protein, which is particularly important for large membrane proteins. Running gels at lower voltage or for longer durations can sometimes improve separation of large membrane proteins.

Is this antibody suitable for detecting endogenous levels of ABCB4, or is it primarily recommended for overexpression systems?

Based on our experience, this antibody is sensitive enough to detect endogenous levels of ABCB4 in cell lines and tissues where the protein is normally expressed. The recombinant nature of the antibody often contributes to high sensitivity and lot-to-lot consistency. While it certainly works well in overexpression systems, its performance with endogenous targets is generally robust. For detection of low endogenous levels, consider optimizing blocking conditions, increasing antibody incubation times (e.g., overnight at 4℃ for primary antibody), and using highly sensitive detection systems.

Mouse Anti-ABCB4 (AA 830-949) Recombinant Antibody (V2-258006) (CBMAB-M0946-FY)

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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

V2-258006

Application

FC, ICC, IF, IHC, WB

Immunogen

Recombinant human MRP3 (multidrug resistance-associated protein 3) (aa830-949)

Specificity

Human

Antibody Isotype

IgG1

Clonality

Monoclonal

Application Notes

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

Application	Note
WB	1:20-1:50
IHC-P	1:20
IHC	1:20
IF(ICC)	1:20-1:50

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

Format

Supernatant

Buffer

0.7% BSA

Preservative

0.09% sodium azide

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.

Epitope

AA 830-949

More Infomation

Target

Full Name

ATP Binding Cassette Subfamily B Member 4

Introduction

The membrane-associated protein encoded by this gene is a member of the superfamily of ATP-binding cassette transporters. ABC proteins transport various molecules across extra- and intra-cellular membranes. ABC genes are divided into seven distinct subfamilies. This protein is a member of the MDR/TAP subfamily. Members of the MDR/TAP subfamily are involved in multidrug resistance as well as antigen presentation. This gene encodes a full transporter and member of the p-glycoprotein family of membrane proteins with phosphatidylcholine as its substrate. The function of this protein has not yet been determined; however, it may involve transport of phospholipids from liver hepatocytes into bile. Alternative splicing of this gene results in several products of undetermined function.

Entrez Gene ID

5244

UniProt ID

P21439

Alternative Names

ATP Binding Cassette Subfamily B Member 4; ATP-Binding Cassette, Sub-Family B (MDR/TAP), Member 4; ATP-Binding Cassette Sub-Family B Member 4; Multidrug Resistance Protein 3; P-Glycoprotein 3; EC 3.6.3.44; MDR3; PGY3; P-Glycoprotein-3/Multiple Drug Resistance-3; Phosphatidylcholine Translocator ABCB4

Function

Energy-dependent phospholipid efflux translocator that acts as a positive regulator of biliary lipid secretion. Functions as a floppase that translocates specifically phosphatidylcholine (PC) from the inner to the outer leaflet of the canalicular membrane bilayer into the canaliculi of hepatocytes. Translocation of PC makes the biliary phospholipids available for extraction into the canaliculi lumen by bile salt mixed micelles and therefore protects the biliary tree from the detergent activity of bile salts. Plays a role in the recruitment of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM) molecules to nonraft membranes and to further enrichment of SM and cholesterol in raft membranes in hepatocytes. Required for proper phospholipid bile formation (By similarity). Indirectly involved in cholesterol efflux activity from hepatocytes into the canalicular lumen in the presence of bile salts in an ATP-dependent manner. Promotes biliary phospholipid secretion as canaliculi-containing vesicles from the canalicular plasma membrane. In cooperation with ATP8B1, functions to protect hepatocytes from the deleterious detergent activity of bile salts. Does not confer multidrug resistance (By similarity).

Biological Process

Bile acid secretion
Cellular response to bile acid
Ceramide translocation
Lipid homeostasis
Lipid metabolic process
Phospholipid translocation
Positive regulation of cholesterol transport
Positive regulation of phospholipid translocation
Positive regulation of phospholipid transport
Regulation of metabolic process
Response to fenofibrate
Transmembrane transport

Cellular Location

Cytoplasm; Cell membrane; Apical cell membrane; Membrane raft; Clathrin-coated vesicle. Localized at the apical canalicular membrane of the epithelial cells lining the lumen of the bile canaliculi and biliary ductules (By similarity). Transported from the Golgi to the apical bile canalicular membrane in a RACK1-dependent manner. Redistributed into pseudocanaliculi formed between cells in a bezafibrate- or PPARA-dependent manner. Localized preferentially in lipid nonraft domains of canalicular plasma membranes.

Involvement in disease

A disorder characterized by early onset of cholestasis that progresses to hepatic fibrosis, cirrhosis, and end-stage liver disease before adulthood.
A liver disorder of pregnancy. It presents during the second or, more commonly, the third trimester of pregnancy with intense pruritus which becomes more severe with advancing gestation and cholestasis. It causes fetal distress, spontaneous premature delivery and intrauterine death. Patients have spontaneous and progressive disappearance of cholestasis after delivery. Cholestasis results from abnormal biliary transport from the liver into the small intestine.
One of the major digestive diseases. Gallstones composed of cholesterol (cholelithiasis) are the common manifestations in western countries. Most people with gallstones, however, remain asymptomatic through their lifetimes.

Topology

Cytoplasmic: 1-50 aa
Helical: 51-73 aa
Extracellular: 74-118 aa
Helical: 119-139 aa
Cytoplasmic: 140-188 aa
Helical: 189-210 aa
Extracellular: 211-217 aa
Helical: 218-238 aa
Cytoplasmic: 239-296 aa
Helical: 297-318 aa
Extracellular: 319-332 aa
Helical: 333-354 aa
Cytoplasmic: 355-711 aa
Helical: 712-732 aa
Extracellular: 733-755 aa
Helical: 756-776 aa
Cytoplasmic: 777-831 aa
Helical: 832-852 aa
Extracellular: 853 aa
Helical: 854-873 aa
Cytoplasmic: 874-933 aa
Helical: 934-956 aa
Extracellular: 957-972 aa
Helical: 973-994 aa
Cytoplasmic: 995-1286 aa

PTM

Phosphorylated. Phosphorylation on Thr-34 is required for PC efflux activity. Phosphorylation occurs on serine and threonine residues in a protein kinase A- or C-dependent manner. May be phosphorylated on Thr-44 and Ser-49.
Glycosylated

Shankar, S., Pande, A., Geetha, T. S., Raichurkar, K., Sakpal, M., Lochan, R., & Asthana, S. (2021). A New Variant of an Old Itch: Novel Missense Variant in ABCB4 Presenting with Intractable Pruritus. Journal of Clinical and Experimental Hepatology.

Kennedy, L., Meadows, V., Kyritsi, K., Pham, L., Kundu, D., Kulkarni, R., ... & Francis, H. (2020). Histamine-2 Receptor Vivo-Morpholino Treatment Ameliorates Large Bile Duct Damage in Mice Deficient in ATP Binding Cassette Subfamily B Member 4 (Abcb4-/-) via Down-Regulation of cAMP/ERK Signaling. The American Journal of Pathology.

Sticova, E., & Jirsa, M. (2020). ABCB4 disease: Many faces of one gene deficiency. Annals of hepatology, 19(2), 126-133.

Sticova, E., Neroldova, M., Kotalova, R., Subhanova, I., & Jirsa, M. (2020). ABCB4 disease mimicking morbus Wilson: A potential diagnostic pitfall. Biomedical Papers, 164(1), 121-125.

Ishizawa, T., Makino, N., Kakizaki, Y., Ando, Y., Matsuda, A., Kobayashi, T., ... & Ueno, Y. (2019). A novel pathogenic variant of ATP-binding cassette subfamily B member 4 causing gallstones in a young adult. Clinical journal of gastroenterology, 12(6), 637-641.

Wen, C., Fu, L., Huang, J., Dai, Y., Wang, B., Xu, G., ... & Zhou, H. (2019). Curcumin reverses doxorubicin resistance via inhibition the efflux function of ABCB4 in doxorubicin‑resistant breast cancer cells. Molecular medicine reports, 19(6), 5162-5168.

Reichert, M. C., & Lammert, F. (2018, November). ABCB4 gene aberrations in human liver disease: an evolving spectrum. In Seminars in liver disease (Vol. 38, No. 04, pp. 299-307). Thieme Medical Publishers.

Huang, J. F., Wen, C. J., Zhao, G. Z., Dai, Y., Li, Y., Wu, L. X., & Zhou, H. H. (2018). Overexpression of ABCB4 contributes to acquired doxorubicin resistance in breast cancer cells in vitro. Cancer chemotherapy and pharmacology, 82(2), 199-210.

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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)
Proteogenomic
Other Protocols

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

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