BIN1 Protein (Transcript Variant 8) (Myc-DYKDDDDK Tag)

Produktnummer ABIN2715270

Dieses Recombinant BIN1-Protein wird in HEK-293 Cells produziert. Es wurde unabhängig validiert.

Kurzübersicht für BIN1 Protein (Transcript Variant 8) (Myc-DYKDDDDK Tag) (ABIN2715270)

Target: BIN1 (Bridging Integrator 1 (BIN1))

Protein-Typ: Recombinant

Spezies: Human

Quelle: HEK-293 Cells

Applikation: Antibody Production (AbP), Standard (STD)

Reinheit: > 80 % as determined by SDS-PAGE and Coomassie blue staining

Produktdetails

Proteineigenschaft: Transcript Variant 8

Aufreinigungstag / Konjugat: Dieses BIN1 Protein ist gelabelt mit Myc-DYKDDDDK Tag.

Produktmerkmale:

• Recombinant human BIN1 / AMPHL (transcript variant 8) protein expressed in HEK293 cells.
• Produced with end-sequenced ORF clone

Anwendungsinformationen

Applikationshinweise: Recombinant human proteins can be used for:
Native antigens for optimized antibody production
Positive controls in ELISA and other antibody assays

Kommentare:

The tag is located at the C-terminal.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Independent Validation (WB)

Validierung #102757 (Western Blotting)

by: Department of Medical Physiology, UMC Utrecht

No.: #102757

Datum: 03.05.2018

Antigen: BIN1

Chargennummer: 10C425

Validierte Anwendung: Western Blotting

Positivkontrolle: BIN1 antibody, endogenous BIN1 in mouse heart lysates and human plasma and heart lysate

Negativkontrolle: none

Bewertung:

Passed. The recombinant BIN1 protein ABIN2715270 works excellently in western blot and appears at the expected MW.

Validierungsbilder

Western blot analysis of human recombinant BIN1 ABIN2715270 (left panel) in comparison to the endogenous murine and human proteins (right panel).

Protokoll

Primärantikörper: rabbit anti-BIN1 antibody (Sigma-Aldrich, HPA003894, lot R04603)

Sekundärantikörper: goat anti-rabbit HRP-conjugated antibody (Bio-Rad Cat, 170-6515)

Full Protocol:

• Lyse half a murine heart or a corresponding piece (in size) of human heart tissue in 250µl of lysis buffer (20mM Tris-HCl pH7.4, 150mM NaCl, 10mM Na₂HPO₄, 1% Triton X-100, 1% Na-deoxycholate, 0.1% SDS, 1mM EDTA, 50mM NaF).
• Determine total protein content of the lysates and human plasma samples using Pierce BCA Protein Assay Kit (ThermoFisher Scientific, 23227, lot SC246925).
• Sample preparation:
• Dilute appropriate amount of lysate in lysis buffer + 5x Laemmli SDS sample buffer (lysis buffer:sample buffer 4:1) to achieve 25µg of protein per 20µl of sample, boil the samples for 5min at 96°C.
  • Dilute appropriate amount of human plasma in lysis buffer + 5x Laemmli SDS sample buffer (lysis buffer:sample buffer 4:1) to achieve 25µg of protein per 20µl of sample, boil the samples for 5min at 96°C.
  • 20ng, 100ng, and 300ng recombinant BIN1 (antibodies-online, ABIN2715270, lot 10C425) were diluted in 20µl ddH₂O + 5x Laemmli SDS sample buffer (ddH₂O:sample buffer 4:1), samples were boiled for5min at 96°C.
• Separate the samples on a freshly cast discontinuous denaturing SDS-PAGE 10% running gel in an electrophoresis chamber (Bio-Rad) for approximately 20min at 75-100V for the stacking and 1h at 100-200V for the running gel.
• Transfer proteins onto a nitrocellulose membrane 0.45µm (Bio-Rad, 1620115, lot A10206645) with a Hoefer TE 77 semi-dry transfer unit (Amersham Biosciences) for 70min at 43mA/gel.
• Verify protein transfer through Ponceau Red staining.
• Block the membrane with blocking buffer (TBST containing 5% BSA) for 1h at RT.
• Incubation with primary rabbit anti-BIN1 antibody (Sigma-Aldrich, HPA003894, lot R04603) diluted 1:500 in blocking buffer for ON at 4°C.
• Wash membrane 3x for 10min with TBST.
• Incubation with secondary goat anti-rabbit HRP-conjugated antibody (Bio-Rad Cat, 170-6515) diluted 1:7000 in blocking buffer for 1-2h at RT.
• Wash membrane 3x for 10min with TBST.
• Reveal protein bands using ECL substrate (GE Healthcare, RPN2232, lot 13601176) on an ChemiDoc XRS + (BioRad) and ImageLab (version 6.0.0 build 25, standard edition. 2017, BioRad laboratories, Inc) software was used to determine best exposure time for optimal visualization of bands.

Anmerkungen:

The BIN1 antibody reveals a protein of the expected MW. ABIN2715270 migrates higher than the endogenous murine and human proteins due to the C-terminal myc- and DYKDDDDK-tags.

Handhabung

Konzentration: 50 μg/mL

Buffer: 25 mM Tris.HCl, pH 7.3, 100 mM glycine, 10 % glycerol.

Lagerung: -80 °C

Informationen zur Lagerung: Store at -80°C. Thaw on ice, aliquot to individual single-use tubes, and then re-freeze immediately. Only 2-3 freeze thaw cycles are recommended.

Antigendetails

Target: BIN1 (Bridging Integrator 1 (BIN1))

Andere Bezeichnung: Bin1,amphl

Hintergrund: This gene encodes several isoforms of a nucleocytoplasmic adaptor protein, one of which was initially identified as a MYC-interacting protein with features of a tumor suppressor. Isoforms that are expressed in the central nervous system may be involved in synaptic vesicle endocytosis and may interact with dynamin, synaptojanin, endophilin, and clathrin. Isoforms that are expressed in muscle and ubiquitously expressed isoforms localize to the cytoplasm and nucleus and activate a caspase-independent apoptotic process. Studies in mouse suggest that this gene plays an important role in cardiac muscle development. Alternate splicing of the gene results in several transcript variants encoding different isoforms. Aberrant splice variants expressed in tumor cell lines have also been described.

Molekulargewicht: 50 kDa

NCBI Accession: NP_004296