TIAM1 Antikörper (AA 1-378)

Produktnummer ABIN1982879

Der Schaf Polyklonal anti-TIAM1 Antikörper wird verwendet zum Nachweis von TIAM1 in Proben von Maus. Er wurde validiert für WB. Der Antikörper wurde zudem unabhängig validiert.

Kurzübersicht für TIAM1 Antikörper (AA 1-378) (ABIN1982879)

Target: TIAM1 (T-Cell Lymphoma Invasion and Metastasis 1 (TIAM1))

Reaktivität: Maus

Wirt: Schaf

Klonalität: Polyklonal

Konjugat: Dieser TIAM1 Antikörper ist unkonjugiert

Applikation: Western Blotting (WB)

Produktdetails anti-TIAM1 Antikörper

Bindungsspezifität: AA 1-378

Produktmerkmale: Polyclonal Sheep anti Mouse TIAM1

Immunogen: AA 1-378

Isotyp: IgG

Anwendungsinformationen

Applikationshinweise: Optimal dilutions should be determined by each laboratory for each application.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Independent Validation (WB)

Validierung #100102 (Western Blotting)

by: Instituto de Parasitología y Biomedicina López-Neyra

No.: #100102

Datum: 23.12.2016

Antigen: TIAM1

Chargennummer: 2016090702

Validierte Anwendung: Western Blotting

Positivkontrolle: primary mouse microglia, MCF-7

Bewertung: Passed. ABIN1982879 specifically recognizes TIAM1 in lysates from human MCF-7 cells.

Validierungsbilder

ABIN1982879 was used to reveal TIAM1 in lysates from human MCF-7 (lane 2) cells and mouse primary mouse microglia (lane 1) as described in the protocol.

Protokoll

Primärantikörper: ABIN1982879

Sekundärantikörper: anti-goat/sheep HRP-conjugated antibodies (DakoCytomation, P0449, lot 00062104)

Full Protocol:

• Primary mouse microglia and human MCF-7 cells are grown in DMEM (Invitrogen), supplemented with 10% foetal bovine serum (Gibco), 10% horse serum and penicillin and streptomycin (Gibco), at 37°C and 5% CO2 in a 12-well plate or in RPMI 1640 (Invitrogen), supplemented with 6% inactivated foetal bovine serum (Gibco), GlutaMax (Gibco), penicillin and streptomycin (Gibco), at 37°C and 5% CO2 in a 10cm dish, respectively.
• Lyse microglia in 25µl per well and MCF-7 cells in 500µl per dish cold lysis buffer (10mM Tris-HCl pH 8.0, 150mM NaCl, 1% Nonidet-P40, 1mM EDTA, 10mM NaF, 1mM Na_3VO4, containing protease inhibitors (Sigma)).
• Determine total protein content of the lysates using Bradford assay (Bio-Rad, 500-0006, lot 111832).
• Denature 30µg total protein for 5min at 95°C in in 20µl Laemmli SDS sample buffer and subsequently separate them on a denaturing, freshly cast 7.5% SDS-PAGE for 1h at 140V.
• Transfer proteins onto PVDF membrane (Pall Life Sciences, 75696G, lot TO3225) with a wet Western blotting system for 80min at 100V.
• Block the membrane with TBST (TBS, 0.1% Tween) containing 5% milk ON at 4°C.
• Incubation with primary sheep anti-TIAM1 antibody (antibodies-online, ABIN1982879, lot 2016090702) diluted 1:500 in TBST containing 5% milk for 1h at RT.
• Wash membrane 5x 5min with TBST.
• Incubation with anti-goat/sheep horseradish peroxidase-conjugated secondary antibodies (DakoCytomation, P0449, lot 00062104) diluted 1:2000 in TBST containing 5% milk for 1h at RT.
• Wash membrane 5x 5min with TBST and 3x 5min with TBS.
• Reveal protein bands using self-made ECL Western Blotting Detection Reagent with Curix RP2 Plus medical X-ray films (Agfa, ENKMV, lot 79040074) on an Agfa- Curix60-developer.

Anmerkungen: The TIAM1 antibody ABIN1982879 reveals a protein of the expected molecular weight of 177kDa in human MCF-7 lysates as verified with a second anti-TIAM1 antibody. However, note that ABIN1982879 did not recognize Tiam1 in primary mouse microglia extracts.

Handhabung

Format: Lyophilized

Rekonstitution: 100 µL of PBS, pH 7.4

Konzentration: 500 µg/mL

Buffer: Lyophilized from a solution containing PBS, pH 7.4

Lagerung: -20 °C

Details zu TIAM1

Target: TIAM1 (T-Cell Lymphoma Invasion and Metastasis 1 (TIAM1))

Andere Bezeichnung: TIAM1

UniProt: Q13009