INPP5E antibody (Inositol Polyphosphate-5-Phosphatase, 72 KDa) (C-Term)

Details for Product anti-INPP5E Antibody No. ABIN2704925, Supplier: Log in to see
Antigen
  • 1200002L24Rik
  • 72kDa
  • mKIAA0123
  • CORS1
  • CPD4
  • JBTS1
  • MORMS
  • PPI5PIV
  • PMPCA
  • inositol polyphosphate-5-phosphatase E
  • Inpp5e
  • INPP5E
Epitope
C-Term
4
3
2
1
Reactivity
Human, Mouse (Murine), Rat (Rattus)
19
10
10
1
1
1
1
1
1
1
1
1
Host
Rabbit
16
3
Clonality
Polyclonal
Application
Western Blotting (WB)
16
7
5
5
2
2
1
1
Options
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'Independent Validation' Badge
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Inpp5e+/+ 4T1 mouse breast carcinoma cell line (transfected with non-targeting Cas9 gRNA), treated with an inducer (unpublished)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 Inpp5eem 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.

Primary Antibody ABIN2704925
Secondary Antibody goat anti-rabbit IgG (H+L)-HRP-conjugated antibody (BioRad, 172-1019, lot 350001898)
Protocol
  • Grow Inpp5e+/+ and Inpp5eem 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  • Treat cells with an “inducer” (unpublished) to increase Inpp5e expression.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.
Validation Images
Western Blotting validation image for anti-Inositol Polyphosphate-5-Phosphatase, 72 KDa (INPP5E) (C-Term) antibody (ABIN2704925) Western blot of 4T1 mouse breast carcinoma cell line extracts (Inpp5e+/+, ...
Immunogen KLH-conjugated synthetic peptide encompassing a sequence within the C-term region of human INPP5E.
Blocking Peptide Blocking peptide for this product available: ABIN2710759
Specificity Recognizes endogenous levels of INPP5E protein.
Characteristics Rabbit polyclonal antibody to INPP5E
Purification The antibody was purified by immunogen affinity chromatography.
Alternative Name INPP5E (INPP5E Antibody Abstract)
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
Research Area Signaling, Metabolism
Application Notes WB (1:500 - 1:1000)
Restrictions For Research Use only
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
Supplier Images
Western Blotting (WB) image for anti-Inositol Polyphosphate-5-Phosphatase, 72 KDa (INPP5E) (C-Term) antibody (ABIN2704925) Western blot analysis of INPP5E expression in HEK293T (A), SP2/0 (B), H9C2 (C) whole ...
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