INPP5E Antikörper (C-Term)

Produktnummer ABIN7298962

Dieser Anti-INPP5E Antikörper ist ein Kaninchen Polyklonal Antikörper zur Detektion von INPP5E in WB. Geeignet für Human, Maus und Ratte. Unabhängig validiert für die Verwendung in Western Blotting.

Kurzübersicht für INPP5E Antikörper (C-Term) (ABIN7298962)

Target: INPP5E (Inositol Polyphosphate-5-Phosphatase, 72 KDa (INPP5E))

Reaktivität: Human, Maus, Ratte

Wirt: Kaninchen

Klonalität: Polyklonal

Konjugat: Dieser INPP5E Antikörper ist unkonjugiert

Applikation: Western Blotting (WB)

Produktdetails anti-INPP5E Antikörper

Bindungsspezifität: C-Term

Spezifität: Recognizes endogenous levels of INPP5E protein.

Produktmerkmale: Rabbit polyclonal antibody to INPP5E

Aufreinigung: The antibody was purified by immunogen affinity chromatography.

Immunogen: KLH-conjugated synthetic peptide encompassing a sequence within the C-term region of human INPP5E.

Anwendungsinformationen

Applikationshinweise: WB (1:500 - 1:1000)

Beschränkungen: Nur für Forschungszwecke einsetzbar

Independent Validation (WB)

Validierung #101129 (Western Blotting)

by: Children's Hospital of Eastern Ontario Research Institute

No.: #101129

Datum: 10.11.2017

Antigen: INPP5E

Chargennummer: CP1D10A

Validierte Anwendung: Western Blotting

Positivkontrolle: Inpp5e^+/+ 4T1 mouse breast carcinoma cell line (transfected with non-targeting Cas9 gRNA), treated with an inducer (unpublished)

Negativkontrolle: Inpp5e^em 4T1 cell line (CRISPR/Cas9 endonuclease mediated Inpp5e knock-out)

Bewertung:

Passed. ABIN2704925 specifically recognizes Inpp5e in murine Inpp5e^+/+ 4T1 cells upon induction aside from other unspecific bands.

Validierungsbilder

Western blot of 4T1 mouse breast carcinoma cell line extracts (Inpp5e+/+, lane 1 and 2) and 4T1 lnpp5e CRISPR/Cas9 knock-out cell extracts (Inpp5eem, lane 3 and 4) with ABIN2704925. A single band appears upon cell treatment with an inducer (unpublished) specifically in the Inpp5e+/+ cells.

Protokoll

Primärantikörper: ABIN2704925

Sekundärantikörper: goat anti-rabbit IgG (H+L)-HRP-conjugated antibody (BioRad, 172-1019, lot 350001898)

Full Protocol:

• Grow Inpp5e^+/+ and Inpp5e^em 4T1 murine cells in HyClone TM RPMI-1640 medium (GE Healthcare Life Science, SH30027.01, lot AC11016269) supplemented with fetal bovine serum (Sigma, F1051-500ML, lot 17D192) and HyClone penicillin – streptomycine solution (GE Healthcare Life Science, SV30010, lot SH40003.01), at 37°C and 5% CO₂ in 3ml on a 6-well plate to 95% confluency.
• Treat cells with an “inducer” (unpublished) to increase Inpp5e expression.
• Lyse cells in 200µl per well of cold RIPA buffer (150mM NaCl, 1.0% IGEPAL CA-630 Sigma, 0.5% sodium deoxycholate, 0.1% SDS, 50mM Tris, pH8.0).
• Determine the total protein content of the lysates using DC Protein Assay (BioRad, 500-0114, lot 200008008).
• Denature 30µg of total protein for 5min at 95°C in 5x Laemmli SDS sample buffer and subsequently separate proteins on a non-/denaturing 10% SDS-polyacrylamide gel in a mini-Protean electrophoresis cell (BioRad) for 90min at 100V.
• Transfer proteins onto 0.2µm nitrocellulose membrane (BioRad, 1620112, lot A10088580) with a MiniTransblot Cell (BioRad, 1703930) system for 60V at 300mA.
• Block the membrane with TBS containing 0.1% Tween 20 (TBST) and 5% skim milk for 30min at RT.
• Incubation with primary rabbit anti-INNP5E antibody (antibodies-online, ABIN2704925, CP1D10A) diluted 1:1000 in TBST containing 5% BSA and 0.05% NaN₃ ON at 4°C.
• Wash membrane 3x with TBST.
• Incubation with secondary goat anti-rabbit IgG (H+L)-HRP-conjugated antibody (BioRad, 172-1019, lot 350001898) diluted 1:5000 in TBST for 1h at RT.
• Wash membrane 5x with TBST.
• Reveal protein bands using Clarity Western ECL Substrate (BioRad, 1705060, lot 102030837) on a ChemiDoc Imaging System (Biorad, 17001401).

Anmerkungen:

• ABIN2704925 reveals a single band in the "wild type" cell line with treatment. This protein band is abrogated in the CRISPR/Cas9 targeted cells and under non-inducing conditions, thus demonstrating immunospecificity of ABIN2704925 for Inpp5e in the mouse.

• We observed a lower molecular weight than the predicted size of 72kDa for Inpp5e. We suspect that this might be a splice isoform. We observed a similar band also with a different anti-INPP5E antibody (Thermo Fisher Scientific, PA5-37119) using similar experiment condition.

• ABIN2704925 also recognizes potentially unspecific bands at various sizes.

Handhabung

Format: Liquid

Buffer: Liquid in 0.42 % Potassium phosphate, 0.87 % Sodium chloride, pH 7.3, 30 % glycerol, and 0.01 % sodium azide.

Konservierungsmittel: Sodium azide

Vorsichtsmaßnahmen: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Lagerung: -20 °C

Informationen zur Lagerung: Shipped at 4°C. Upon delivery aliquot and store at -20°C for one year. Avoid freeze/thaw cycles.

Haltbarkeit: 12 months

Details zu INPP5E

Target: INPP5E (Inositol Polyphosphate-5-Phosphatase, 72 KDa (INPP5E))

Andere Bezeichnung: INPP5E

Hintergrund: 72 kDa inositol polyphosphate 5-phosphatase, Phosphatidylinositol 4,5-bisphosphate 5-phosphatase, Phosphatidylinositol polyphosphate 5-phosphatase type IV

Gen-ID: 56623, 64436

UniProt: Q9NRR6, Q9JII1, Q9WVR1