ET1702-03

Src Recombinant Rabbit Monoclonal Antibody [JF0947]

cat.: ET1702-03

Product Type:	Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity:	Human, Mouse, Rat
Applications:	WB, IF-Cell, IF-Tissue, IHC-P, IP, FC
Clonality:	Monoclonal
Clone number:	JF0947

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: 60 kDa
Isotype:	IgG
Immunogen:	Synthetic peptide within human Src aa 20-60.
Positive control:	SK-OV-3 cell lysate, A549 cell lysate, NIH/3T3 cell lysate, 4T1 cell lysate, PC-12 cell lysate, C6 cell lysate, COS-1 cell lysate, SK-OV-3, NIH/3T3, PC-12, human kidney tissue, mouse kidney tissue, rat kidney tissue, SK-OV-3 Y.
Subcellular location:	Cell membrane, Mitochondrion inner membrane, Nucleus, Cytoplasm.
Recommended Dilutions: WB IF-Cell IF-Tissue IHC-P IP FC	1:1,000-1:5,000 1:100-1:500 1:100-1:500 1:20-1:1,000 1/2,000 1:50-1:100
Uniprot #:	SwissProt: P12931 Human \| P05480 Mouse \| Q9WUD9 Rat
Alternative names:	ASV Avian sarcoma virus c SRC CDNA FLJ14219 fis clone NT2RP3003800 highly similar to Rattus norvegicus tyrosine protein kinase pp60 c src mRNA cSrc EC 2.7.10.2 Neuronal CSRC tyrosine specific protein kinase Neuronal SRC Oncogene SRC OTTHUMP00000174476 OTTHUMP00000174477 p60 Src p60-Src p60Src pp60c src pp60c-src pp60csrc Proto oncogene tyrosine protein kinase Src Proto-oncogene c-Src Proto-oncogene tyrosine-protein kinase Src Protooncogene SRC Protooncogene SRC Rous sarcoma Src SRC Oncogene SRC proto oncogene non receptor tyrosine kinase SRC_HUMAN SRC1 Tyrosine kinase pp60c src Tyrosine protein kinase SRC 1 Tyrosine protein kinase SRC1 v src avian sarcoma (Schmidt Ruppin A2) viral oncogene homolog V src sarcoma (Schmidt Ruppin A 2) viral oncogene homolog (avian) v src sarcoma (Schmidt Ruppin A 2) viral oncogene homolog avian

Images

Fig1: Western blot analysis of Src on different lysates with Rabbit anti-Src antibody (ET1702-03) at 1/1,000 dilution.

Lane 1: SK-OV-3 cell lysate, 20 µg/Lane
Lane 2: A549 cell lysate, 20 µg/Lane
Lane 3: NIH/3T3 cell lysate, 20 µg/Lane
Lane 4: 4T1 cell lysate, 20 µg/Lane
Lane 5: PC-12 cell lysate, 20 µg/Lane
Lane 6: C6 cell lysate, 20 µg/Lane
Lane 7: COS-1 cell lysate, 20 µg/Lane

Lysates/proteins at 20 µg/Lane.

Predicted band size: 60 kDa
Observed band size: 60 kDa

Exposure time: 16 seconds;
4-20% 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 (ET1702-03) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Fig2: All lanes: Western blot analysis of Src with anti-Src antibody [JF0947] (ET1702-03) at 1:1,000 dilution.

Lane 1: Wild-type HCT116 whole cell lysate (20 µg).
Lane 2: Src knockout HCT116 whole cell lysate (20 µg).

ET1702-03 was shown to specifically react with Src in wild-type HCT116 cells. No band was observed when Src knockout sample was tested. Wild-type and Src knockout samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary antibody (ET1702-03, 1/1,000) and Loading control antibody (Rabbit anti-GAPDH , ET1601-4, 1/10,000) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG-HRP Secondary Antibody (HA1001) at 1:200,000 dilution was used for 1 hour at room temperature.

Fig3: Immunocytochemistry analysis of SK-OV-3 cells labeling Src with Rabbit anti-Src antibody (ET1702-03) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Src antibody (ET1702-03) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Fig4: Immunocytochemistry analysis of NIH/3T3 cells labeling Src with Rabbit anti-Src antibody (ET1702-03) at 1/50 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Src antibody (ET1702-03) at 1/50 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

	Fig5: Immunocytochemistry analysis of PC-12 cells labeling Src with Rabbit anti-Src antibody (ET1702-03) at 1/100 dilution. Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Src antibody (ET1702-03) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
	Fig6: Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-Src antibody (ET1702-03) 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 (ET1702-03) 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.
	Fig7: Immunohistochemical analysis of paraffin-embedded mouse kidney tissue with Rabbit anti-Src antibody (ET1702-03) at 1/1,000 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 (ET1702-03) at 1/1,000 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.

	Fig8: Immunohistochemical analysis of paraffin-embedded rat kidney tissue with Rabbit anti-Src antibody (ET1702-03) 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 (ET1702-03) 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.
	Fig9: Src was immunoprecipitated in 0.2mg A549 cell lysate with (ET1702-03) at 2 µg/25 µl agarose. Western blot was performed from the immunoprecipitate using (ET1702-03) at 1/2,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature. Lane 1: A549 cell lysate (input) Lane 2: (ET1702-03) IP in A549 cell lysate Lane 3: Rabbit IgG instead of (ET1702-03) in A549 cell lysate Blocking/Dilution buffer: 5% NFDM/TBST Exposure time: 6s
	Fig10: Flow cytometric analysis of SK-OV-3 Y cells labeling Src. Cells were fixed and permeabilized. Then stained with the primary antibody (ET1702-03, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

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