SPNS2 antibody (N-Term)

Catalog No. ABIN2786494

The Rabbit Polyclonal anti-SPNS2 antibody has been validated for WB and has been independently validated. It is suitable to detect SPNS2 in samples from Human, Mouse, Rat, Pig and Rabbit.

Quick Overview for SPNS2 antibody (N-Term) (ABIN2786494)

Target: SPNS2 (Spinster Homolog 2 (SPNS2))

Reactivity: Human, Mouse, Rat, Pig, Rabbit

Host: Rabbit

Clonality: Polyclonal

Conjugate: This SPNS2 antibody is un-conjugated

Application: Western Blotting (WB)

Product Details anti-SPNS2 Antibody

Binding Specificity: N-Term

Sequence: PPGTPGTPGC AATAKGPGAQ QPKPASLGRG RGAAAAILSL GNVLNYLDRY

Predicted Reactivity: Human: 100%, Mouse: 85%, Pig: 100%, Rabbit: 92%, Rat: 93%

Characteristics: This is a rabbit polyclonal antibody against SPNS2. It was validated on Western Blot using a cell lysate as a positive control.

Purification: Affinity Purified

Immunogen: The immunogen is a synthetic peptide directed towards the N terminal region of human SPNS2

Application Details

Application Notes: Optimal working dilutions should be determined experimentally by the investigator.

Comment:

Antigen size: 549 AA

Restrictions: For Research Use only

Independent Validation (WB)

Validation #101829 (Western Blotting)

by: Puissant lab, Institute of Hematology, Inserm U944, CNRS UMR 7212

No.: #101829

Date: 02/01/2018

Antigen: SPNS2

Lot Number: QC66273-42972

Method validated: Western Blotting

Positive Control: Baf-3 cells/ MLL-AF9 murine primary cell model

Negative Control: Baf-3 or MLL-AF9 murine primary cell model silenced for SPNS2 gene with 2 different shRNA

Notes:

Passed. ABIN2786494 specifically recognizes SPNS2 in murine Baf-3 cells or MLL-AF9 primary cell model.

Validation Images

Western blot analysis of SPNS2 expression in mouse Baf-3 (left) and MLL-AF9 cells (right) using ABIN2786494. Cells were transduced either with a control shRNA (control shRNA) or one of two SPNS2 shRNA (shSPNS2_1 and shSPNS2_5).

Full Methods

Primary Antibody: ABIN2786494

Secondary Antibody: mouse anti-rabbit HRP conjugated antibody (GE Healthcare, NA9340V)

Full Protocol:

• Grow Baf-3 or MLL-AF9 cells in RPMI 1640 1x (Gibco, 21875-059) supplemented with 10% FBS (Sigma-Aldrich, F6178, lot 16G148) and 1x Penicillin/Streptomycin (Gibco, 15140-122), at 37°C and 5% CO₂ in 10ml on a flask to 70% confluency.
• Transduce 3x10⁶ cells with 35µl of concentrated retrovirus, spin-infection (3h at 37°C to 1200 rpm).
• Grow cells for 3d.
• Induce and select cells.
• Grow cells for 5d.
• Sort and collect cells (doxycycline).
• Lyse 7x10⁵ cells in 25µl per well in cold SDS Lysis Buffer (Cell Signaling).
• Determine total protein content of the lysates using Bradford assay (Bio-Rad, 5000006).
• Denature 20µg of total protein for 7min at 95°C in 6µl Laemmli SDS sample buffer and subsequently separate them on a denaturing NuPAGE 4-12% Bis-Tris Protein Gels (Invitrogen,NP0321BOX ) in an ELECTROPHORESIS CHAMBER for 2h at 150V.
• Transfer proteins onto PVDF membrane (Millipore) with a Western blotting system for 1h 40min at 300mA.
• Block the membrane with blocking buffer (2.75% gelatine, 30% BSA, 10mM Tris, NaCl, EDTA, tween 20) for 1h at RT.
• Incubation with rabbit anti-SPNS2 antibody (antibodies-online, ABIN2786494, lot QC66273-42972) diluted 1:500 in blocking buffer for 16h at 4°C.
• Wash membrane 3x for 10min with tween TBS buffer.
• Incubation with secondary mouse anti-rabbit HRP conjugated antibody (GE Healthcare, NA9340V) diluted 1:5000 in blocking buffer for 1h at RT.
• Wash membrane 3x for 10min with TBST buffer.
• Reveal protein bands using Pierce ECL Western Blotting Substrate (ThermoFisher Scientific, 32106) on a Li-Cor imaging machine.

Experimental Notes:

• The SPNS2 antibody ABIN2786494 reveals a protein of the expected molecular weight for SPNS2 in lysates of murine Baf-3 cells or MLL-AF9 primary cell model.

• ABIN2786494 does show some unspecific binding but the SPNS2 band itself is clear and only visible in the positive but not the negative controls.

Handling

Format: Liquid

Concentration: Lot specific

Buffer: Liquid. Purified antibody supplied in 1x PBS buffer with 0.09 % (w/v) sodium azide and 2 % sucrose.

Preservative: Sodium azide

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

Handling Advice: Avoid repeated freeze-thaw cycles.

Storage: -20 °C

Storage Comment: For short term use, store at 2-8°C up to 1 week. For long term storage, store at -20°C in small aliquots to prevent freeze-thaw cycles.

Target Details for SPNS2

Target: SPNS2 (Spinster Homolog 2 (SPNS2))

Alternative Name: SPNS2

Background: SPNS2 is the sphingolipid transporter required for migration of myocardial precursors. SPNS2 transports sphingosine 1-phosphate (S1P), a secreted lipid mediator that plays critical roles in cardiovascular, immunological, and neural development and function. SPNS2 mediates the export of S1P from cells in the extraembryonic yolk syncytial layer (YSL), thereby regulating myocardial precursor migration.
Alias Symbols: -
Protein Size: 549

Molecular Weight: 60 kDa

Gene ID: 124976

NCBI Accession: NM_001124758, NP_001118230

UniProt: Q8IVW8