ENTPD5 antibody (Center)

Catalog No. ABIN2856876

This anti-ENTPD5 antibody is a Rabbit Polyclonal antibody detecting ENTPD5 in WB and IHC (p). Suitable for Human. Independently validated for use in Western Blotting.

Quick Overview for ENTPD5 antibody (Center) (ABIN2856876)

Target: ENTPD5 (Ectonucleoside Triphosphate diphosphohydrolase 5 (ENTPD5))

Reactivity: Human

Host: Rabbit

Clonality: Polyclonal

Conjugate: This ENTPD5 antibody is un-conjugated

Application: Western Blotting (WB), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p))

Product Details anti-ENTPD5 Antibody

Binding Specificity: Center

Characteristics: Rabbit polyclonal antibody to ENTPD5(CD39L4) (ectonucleoside triphosphate diphosphohydrolase 5)
ENTPD5 antibody [N1N3]

Purification: Purified by antigen-affinity chromatography.

Immunogen: Recombinant protein encompassing a sequence within the center region of human ENTPD5.

Application Details

Application Notes: WB: 1:500-1:3000, IHC-P: 1:100-1:1000.
Not yet tested in other applications.
Optimal working dilutions should be determined experimentally by the end user.

Restrictions: For Research Use only

Independent Validation (WB)

Validation #101762 (Western Blotting)

by: Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago

No.: #101762

Date: 11/02/2017

Antigen: ENTPD5

Lot Number: 40128

Method validated: Western Blotting

Positive Control: Tet-inducible ENTPD5 LNCaP cells, Tet-inducible ENTPD5 BPH1 cells

Negative Control: BPH1 cells

Notes:

Passed. ABIN2856876 specifically recognizes ENTPD5 in Tet-inducible ENTPD5 LNCaP cells and Tet-inducible ENTPD5 BPH1 cells.

Validation Images

A Western blot detection of ENTPD5 in lysates of untransfected LNCaP (1) and BPH1 (4) cells, doxycline-induced GFP-LNCaP (2), ENTP5-LNCaP (3), and BPH1-ENTPD5 cells (5) using ABIN2856876 (A) or a reference antibody (B).

Full Methods

Primary Antibody: ABIN2856876

Secondary Antibody: HRP-conjugated goat-anti rabbit antibody (ThermoFisher, A16104, lot 42-28-042114)

Full Protocol:

• Grow
• • LNCaP cells in RPMI 1640 1x with L-glutamine medium (Corning Cellgro, 10-040-CV, lot 11717002) supplemented with 10% FBS (GE Healthcare, SH30910.03, lot AC10225544) and penicillin-streptomycin (Mediatech, MT30002CI, lot 30002292) at 37°C and 5% CO₂ in 5ml on a tissue culture dish to 1.5x10⁶ cells/ml.
  • Tet-inducible GFP-LNCaP cells in RPMI 1640 1x with L-glutamine medium supplemented with 10% FBS, penicillin-streptomycin, and 1µg/ml Doxycycline hyclate (Sigma Aldrich, D9891, 056M4029V) at 37°C and 5% CO₂ in 5ml on a tissue culture dish to 1.5x10⁶ cells/ml.
  • Tet-inducible ENTPD5-LNCaP cells in RPMI 1640 1x with L-glutamine medium and 1µg/mL Doxycycline hyclate at 37°C and 5% CO₂ in 5ml on a tissue culture dish to 1.5x10⁶ cells/ml.
  • BPH1 cells in RPMI 1640 1x with L-glutamine medium supplemented with 10% FBS and penicillin-streptomycin at 37°C and 5% CO₂ in 5ml on a tissue culture dish to 1.5x10⁶ cells/ml.
  • Tet-inducible ENTPD5-BPH1 cells in RPMI 1640 1x with L-glutamine medium and 1µg/ml Doxycycline hyclate (Sigma Aldrich, D9891) at 37°C and 5% CO₂ in 5ml on a tissue culture dish to 1.5x10⁶ cells/ml.
• Collect and resuspend cells in 50µl lysis buffer (20mM HEPES pH7.4, 1% Triton X-100, 150mM NaCl, 1mM EGTA, 1mM EDTA, 10mM sodium pyrophosphate, 100mM NaF, 5mM iodoacetic acid, 20nM okadeic acid, 0.2mM PMSF, and 1 tablet/10mL Pierce Protease Inhibitor mini Tablet (ThermoFisher, 88665, Lot RC2161121)).
• Lyse cells by 5 rounds of freeze/thaw cycles: 60sec in LN2, 30sec in a 37°C water bath, 15sec vortex, 60sec on ice.
• Spin lysed cells at 10000xg for 5min at 4°C.
• Recover the supernatant and measure protein concentration with Bradford reagent (Bio-Rad, 5000006).
• Denature 25μg total proteins of each sample for 5min at 95°C in Laemmli SDS sample buffer and subsequently separate them on two separate 4-12% Express Plus Page Gels (GenScript, M41215, Lot:C30251704) for 40min at 160V in an electrophoresis chamber (BioRad).
• Transfer proteins onto PVDF membrane (Millipore, IPFL00010, Lot K0MA0558B) with a wet blotting system (Bio-Rad) for 75min at 115V at 4°C.
• Block the membrane with 5% non-fat dry milk in TBSTween (0.1% v/v) for 1h at RT.
• Incubation the membranes with primary
• • rabbit anti-ENTPD5 antibody (antibodies-online, ABIN2856876, Lot: 40128) or
  • rabbit anti-ENTPD5 antibody (Sigma, HPA002927 Lot: A40077)
• diluted 1:500 in 5% non-fat dry milk in TBST at for 16h at 4°C.
• Wash the membrane 5x for 5min in TBST.
• Incubate the membrane with secondary HRP-conjugated goat-anti rabbit antibody (ThermoFisher, A16104, lot 42-28-042114) diluted 1:3000 in 5% non-fat dry milk in TBST for 1h at RT.
• Wash membrane 5x for 5min with PBST.
• Visualize protein bands using SuperSignal West Pico Chemiluminescent Substrate (ThermoScientific, 34087, Lot SC246874) on CL-X Posure Film (ThermoScientific, 34091).

Experimental Notes:

• The ENTPD5 antibody ABIN2856876 detects a single band around expected molecular weight (47kDa). The intensity of this band becomes more intense upon ENTPD5 overexpression and disappears in the negative control cell line.

• ABIN2856876 also reveals fewer non-specific bands than the control antibody. We recommend this antibody for detecting ENTPD5. The positive control antibody performed as predicted with showing increased expression in the overexpression cells and no expression in the negative control BPH1 cell line.

Handling

Format: Liquid

Concentration: 1 mg/mL

Buffer: 0.1M Tris, 0.1M Glycine, 10 % Glycerol ( pH 7).
0.01 % Thimerosal was added as a preservative.

Preservative: Thimerosal (Merthiolate)

Precaution of Use: This product contains Thimerosal (Merthiolate): a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Storage: -20 °C

Storage Comment: Keep as concentrated solution. Aliquot and store at -20°C or below.
Avoid multiple freeze-thaw cycles.

Target Details for ENTPD5

Target: ENTPD5 (Ectonucleoside Triphosphate diphosphohydrolase 5 (ENTPD5))

Alternative Name: ENTPD5

Gene ID: 957