INPP5E antibody (C-Term)

Catalog No. ABIN7298962

This anti-INPP5E antibody is a Rabbit Polyclonal antibody detecting INPP5E in WB. Suitable for Human, Mouse, Rat. Independently validated for use in Western Blotting.

Quick Overview for INPP5E antibody (C-Term) (ABIN7298962)

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

Reactivity: Human, Mouse, Rat

Host: Rabbit

Clonality: Polyclonal

Conjugate: This INPP5E antibody is un-conjugated

Application: Western Blotting (WB)

Product Details anti-INPP5E Antibody

Binding Specificity: C-Term

Specificity: Recognizes endogenous levels of INPP5E protein.

Characteristics: Rabbit polyclonal antibody to INPP5E

Purification: The antibody was purified by immunogen affinity chromatography.

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

Application Details

Application Notes: WB (1:500 - 1:1000)

Restrictions: For Research Use only

Independent Validation (WB)

Validation #101129 (Western Blotting)

by: Children's Hospital of Eastern Ontario Research Institute

No.: #101129

Date: 11/10/2017

Antigen: INPP5E

Lot Number: CP1D10A

Method validated: Western Blotting

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Inpp5e^+/+ 4T1 mouse breast carcinoma cell line (transfected with non-targeting Cas9 gRNA), treated with an inducer (unpublished)

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

Notes:

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

Validation Images

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 A single band appears upon cell treatment with an inducer (unpublished) specifically in the Inpp5e+/+ cells.

Full Methods

Primary Antibody: ABIN2704925

Secondary Antibody: 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• Treat cells with an “inducer” (unpublished) to increase Inpp5e expression.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).

Experimental Notes:

• 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.

Handling

Format: Liquid

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

Preservative: Sodium azide

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

Storage: -20 °C

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

Expiry Date: 12 months

Target Details for INPP5E

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

Alternative Name: INPP5E

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

Gene ID: 56623, 64436

UniProt: Q9NRR6, Q9JII1, Q9WVR1