ET1702-43

NCAM1 Recombinant Rabbit Monoclonal Antibody [JF1021]

cat.: ET1702-43

Product Type:	Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity:	Human, Zebrafish
Applications:	WB, IF-Cell, IF-Tissue, IHC-P, FC, IP, mIHC
Clonality:	Monoclonal
Clone number:	JF1021

Form:	Liquid
Storage condition:	Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
Storage buffer:	1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration:	1ug/ul
Purification:	Protein A affinity purified.
Molecular weight:	Predicted band size: 95 kDa
Isotype:	IgG
Immunogen:	Synthetic peptide within C-terminal human NCAM1.
Positive control:	SH-SY5Y cell lysates, human brain tissue lysates, A549, SH-SY5Y, human tonsil tissue, zebrafish tissue, human kidney tissue, human small lell lung cancer.
Subcellular location:	Cell membrane, Secreted.
Recommended Dilutions: WB IF-Cell IF-Tissue IHC-P FC IP mIHC	1:1,000-1:5,000 1:50-1:200 1:50-1:200 1:50-1:200 1:50-1:100 Use at an assay dependent concentration. 1:1,000
Uniprot #:	SwissProt: P13591 Human
Alternative names:	antigen MSK39 identified by monoclonal 5.1H11 antigen recognized by monoclonal 5.1H11 CD56 cell adhesion molecule, neural, 1 MSK 39 MSK39 N-CAM-1 NCAM 1 NCAM NCAM C NCAM-1 NCAM1 NCAM1_HUMAN NCAMC Neural cell adhesion molecule 1 Neural cell adhesion molecule NCAM OTTHUMP00000235666

Images

Fig1: Western blot analysis of NCAM1 on SH-SY5Y cell lysates with Rabbit anti-NCAM1 antibody (ET1702-43) at 1/500 dilution.

Lysates/proteins at 10 µg/Lane.

Predicted band size: 95 kDa
Observed band size: 120/140 kDa

Exposure time: 2 minutes;

6% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1702-43) at 1/500 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:300,000 dilution was used for 1 hour at room temperature.

Fig2: Western blot analysis of NCAM1 on human brain tissue lysates with Rabbit anti-NCAM1 antibody (ET1702-43) at 1/2,000 dilution.

Lysates/proteins at 20 µg/Lane.

Predicted band size: 95 kDa
Observed band size: 140/180 kDa

Exposure time: 30 seconds;

6% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1702-43) at 1/2,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:300,000 dilution was used for 1 hour at room temperature.

	Fig3: Fluorescence multiplex immunohistochemical analysis of Tertiary Lymphoid Structures in Human Small Cell Lung Cancer (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-CD20 (HA721138, green), anti-PD-L1 (HA721176, cyan), anti-CD56 (ET1702-43, magenta) and anti-CD3 (HA720082, yellow) on tertiary lymphoid structures. Panel B: anti- CD20 stained on B cells. Panel C: anti-PD-L1 stained on dendritic cells and macrophages cells. Panel D: anti-CD56 stained on NKT cells. Panel E: anti-CD3 stained on T cells. HRP Conjugated UltraPolymer Goat Polyclonal Antibody HA1119/HA1120 was used as a secondary antibody. The immunostaining was performed with the Sequential Immuno-staining Kit (IRISKit™MH010101, www.luminiris.cn). The section was incubated in four rounds of staining: in the order of HA721138 (1/1,500 dilution), HA721176 (1/1,000 dilution), ET1702-43 (1/1,000 dilution), and HA720082 (1/500 dilution) for 20 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 30 mins at 95℃. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Olympus VS200 Slide Scanner.
	Fig4: Fluorescence multiplex immunohistochemical analysis of human colon carcinoma (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-CD68 (EM1901-95, Yellow) and anti-CD56 (ET1702-43, Red) on human colon carcinoma. HRP Conjugated UltraPolymer Goat Polyclonal Antibody HA1119/HA1120 was used as a secondary antibody. The immunostaining was performed with the Sequential Immuno-staining Kit (IRISKit™MH010101, www.luminiris.cn). The section was incubated in two rounds of staining: in the order of EM1901-95 (1/3,000 dilution) and ET1702-43 (1/1,000 dilution) for 20 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 30 mins at 95℃. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Zeiss Observer 7 Inverted Fluorescence Microscope.
	Fig5: ICC staining of NCAM1 in A549 cells (red). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1702-43, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®594 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).

	Fig6: ICC staining of NCAM1 in SH-SY5Y cells (red). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (ET1702-43, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®594 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
	Fig7: Immunohistochemical analysis of paraffin-embedded human tonsil tissue using anti-NCAM1 antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1702-43, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
	Fig8: Immunohistochemical analysis of paraffin-embedded zebrafish tissue using anti-NCAM antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1702-43, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
	Fig9: Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-NCAM1 antibody (ET1702-43) at 1/200 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1702-43) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Fig10: Flow cytometric analysis of NCAM1 was done on SH-SY5Y cells. The cells were fixed, permeabilized and stained with the primary antibody (ET1702-43, 1/50) (red). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor 488-conjugated Goat anti-Rabbit IgG Secondary antibody at 1/1000 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

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