SIGLEC5 antibody

Catalog No. ABIN5541399

The Rat Monoclonal anti-SIGLEC5 antibody has been validated for IP and FACS. It is suitable to detect SIGLEC5 in samples from Mouse.

Quick Overview for SIGLEC5 antibody (ABIN5541399)

Target: SIGLEC5 (Sialic Acid Binding Ig-Like Lectin 5 (SIGLEC5))

Reactivity: Mouse

Host: Rat

Clonality: Monoclonal

Conjugate: This SIGLEC5 antibody is un-conjugated

Application: Immunoprecipitation (IP), Flow Cytometry (FACS)

Clone: 8D2

Product Details anti-SIGLEC5 Antibody

Specificity: This antibody reacts with mouse SiglecE.

Purification: Protein G agarose

Immunogen: Mouse SiglecE transfected L cell

Isotype: IgG2b

Application Details

Application Notes: Immunoprecipitation: 2 μg/300 μL of cell extract from 5x10 6 cells. Flow Cytometry: 5-10 μg/mL (final concentration). For details see protocol below. Not recommended for Western blot.

Protocol: Immunoprecipitation 1) Wash the Biotin-labeled transfectant cells 3 times with PBS and suspend with 10 volume of cold Lysis buffer (50 mM Tris-HCl pH 7.2, 250 mM NaCl, 0.1 % NP-40, 2 mM EDTA, 10 % glycerol) containing appropriate protease inhibitors. Incubate it at 4 o C with rotating for 30 minutes, then sonicate briefly (up to 10 seconds). 2) Centrifuge the tube at 12,000 x g for 10 minutes at 4 o C and transfer the supernatant to another tube. 3) Add primary antibody as suggested in the APPLICATIONS into 200 μ L of the supernatant. Mix well and incubate with gentle agitation for 30-120 minutes at 4 o C. Add 30 μ L of 50 % protein G agarose beads resuspended in the cold Lysis buffer. Mix well and incubate with gentle agitation for 60 minutes at 4 o C. 4) Wash the beads 3-5 times with the cold Lysis buffer (centrifuge the tube at 2,500 x g for 10 seconds). 5) Resuspend the beads in 20 μ L of Laemmli's sample buffer and boil the samples for 2 minutes and centrifuge. Load 10 μ L of the sample per lane in a 1 mm thick SDS-polyacrylamide gel for electrophoresis. 6) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm 2 for 1 hour in a semi-dry transfer system (Transfer Buffer: 25 mM Tris, 190 mM glycine, 20 % MeOH). See the manufacture's manual for precise transfer procedure. 7) To reduce nonspecific binding, soak the membrane in 10 % skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature, or overnight at 4 o C. 8) Incubate the membrane with the 1:10,000 HRP-conjugated streptavidin (MBL code no.284) diluted with 1 % skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature. 9) Wash the membrane with PBS (5 minutes x 6 times). 10) Wipe excess buffer from the membrane, then incubate it with appropriate chemiluminescence reagents for 1 minute. Remove extra reagent from the membrane by dabbing with a paper towel, and seal it in plastic wrap. 11) Expose to X-ray film in a dark room for 5 minutes. Develop the film as usual. The conditions for exposure and development may vary. Positive control for immunop recipitation transfectant Flow cytometric analysis for floating cells Protocol 1 We usually use Fisher tubes or equivalents as reaction tubes for all steps described below. 1) Wash the cells 3 times with washing buffer [PBS containing 2 % fetal calf serum (FCS) and 0.1 % NaN 3]. 2) Resuspend the cells with washing buffer (5x10 6 cells/mL). 3) Add 50 μ L of the cell suspension into each tube, and centrifuge at 500 x g for 1 minute at room temperature (20~25 o C). Remove supernatant by careful aspiration. 4) Add 10 μ L of normal goat serum containing 1 mg/mL normal human IgG and 0.1 % NaN 3 to the cell pellet after tapping. Mix well and incubate for 5 minutes at room temperature (20~25 o C). 5) Add 30 μL of the anti-mouse SiglecE monoclonal antibody (8D2) (5-10 μ g/mL) diluted with the washing buffer. Mix well and incubate for 30 minutes at room temperature (20~25 o C). 6) Add 1 mL of the washing buffer followed by centrifugation at 500 x g for 1 minute at room temperature (20~25 o C). Remove supernatant by careful aspiration. 7) Add 20 μL of 1:100 FITC conjugated anti-rat IgG diluted with the washing buffer. Mix well and incubate for 15 minutes at room temperature (20~25 o C). 8) Add 1 mL of the washing buffer followed by centrifugation at 500 x g for 1 minute at room temperature (20~25 o C). Remove supernatant by careful aspiration. 9) Add 20 μL of 1:50 PE conjugated anti-mouse CD11c diluted with the washing buffer. Mix well and incubate for 15 minutes at room temperature (20~25 o C). 10) Add 1 mL of the washing buffer followed by centrifugation at 500 x g for 1 minute at room temperature (20~25 o C). Remove supernatant by careful aspiration. 11) Resuspend the cells with 500 μ L of the washing buffer and analyze by a flow cytometer. Positive control for flow cytometry mouse splenocyte Protocol 2 2) Resuspend the cells with PBS containing 25 % normal goat serum, 1 mg/mL normal human IgG and 0.1 % NaN 3 (5x10 6 cells/mL). 3) Add 30 μ L of the anti-mouse SiglecE monoclonal antibody (8D2) (50 μ g/mL) diluted with the washing buffer into each tube. 4) Add 50 μ L of the cell suspension into each tube. Mix well and incubate for 30 minutes at room temperature (20~25 o C). 5) Add 1 mL of the washing buffer followed by centrifugation at 500 x g for 1 minute at room temperature (20~25 o C). Remove supernatant by careful aspiration. 6) Resuspend the cells with 50 μ L of the washing buffer. 7) Add 30 μL of 1:100 FITC conjugated anti-rat IgG (Pharmingen: code no.554016) diluted with the washing buffer into each tube. Mix well and incubate for 15 minutes at room temperature (20~25 o C).

Restrictions: For Research Use only

Handling

Format: Liquid

Buffer: PBS containing 50 % glycerol, pH 7.2. Contains no preservatives.

Preservative: Without preservative

Storage: -20 °C

Storage Comment: Upon receipt, store undiluted (in aliquots) at -20°C. Avoid repeated freezing and thawing. Shelf life: One year from despatch.

Target Details for SIGLEC5

Target: SIGLEC5 (Sialic Acid Binding Ig-Like Lectin 5 (SIGLEC5))

Alternative Name: cd170,siglec5

Background: LecE (sialic acid-binding immunoglobulin-like lectin E/SigLec5/CD170) is a putative adhesion molecule that mediates sialic-acid dependent binding to cells. SiglecE is expressed on neutrophil and monocyte popul ations, both in the blood and bone marrow, as a dimeric, disulfide linked, 140 kDa type I membrane protein containing cytoplasmic immune receptor-based tyrosine signaling motifs. SiglecE recruits the SH2-domain-containing protein tyrosine phosphatases SHP-1 and SHP-2, which block signal transduction through dephosphorylation of signaling molecules. Thus SiglecE acts as an inhibitory receptor for ligand induced tyrosine phosphorylation.

UniProt: O15389