ET1610-43

Cytokeratin 5 Recombinant Rabbit Monoclonal Antibody [SC62-04]

cat.: ET1610-43

Product Type:	Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity:	Human, Mouse, Rat
Applications:	WB, IHC-P, IF-Tissue, IF-Cell, FC
Clonality:	Monoclonal
Clone number:	SC62-04

Form:	Liquid
Storage condition:	Shipped at 4℃. Store at +4℃ short term (1-2 weeks). It is recommended to aliquot into single-use upon delivery. Store at -20℃ long term.
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: 62 kDa
Isotype:	IgG
Immunogen:	Synthetic peptide within human Cytokeratin 5 aa 70-110.
Positive control:	A431, A431 cell lysate, MDA-MB-468 cell lysate, MCF7 cell lysate, mouse skin tissue lysate, rat skin tissue lysate, human skin tissue, human tonsil tissue, human lung squamous cell carcinoma tissue, human breast carcinoma tissue.
Subcellular location:	Cytoplasm.
Recommended Dilutions: WB IHC-P IF-Tissue IF-Cell FC	1:5,000 1:500-1:2,000 1:200 1:500 1:1,000
Uniprot #:	SwissProt: P13647 Human \| Q922U2 Mouse \| Q6P6Q2 Rat
Alternative names:	58 kDa cytokeratin CK-5 CK5 Cytokeratin-5 Cytokeratin5 DDD DDD1 EBS2 epidermolysis bullosa simplex 2 Dowling-Meara/Kobner/Weber-Cockayne types K2C5_HUMAN K5 keratin 5 (epidermolysis bullosa simplex, Dowling-Meara/Kobner/Weber-Cockayne types) Keratin 5 Keratin keratin complex 2, basic, gene 5 keratin, type II cytoskeletal 5 Keratin-5 Keratin5 KRT 5 Krt5 KRT5A type II cytoskeletal 5 Type-II keratin Kb5

Images

Fig1: Immunocytochemistry analysis of A431 cells labeling Cytokeratin 5 with Rabbit anti-Cytokeratin 5 antibody (ET1610-43) at 1/500 dilution and competitor's antibody 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-Cytokeratin 5 antibody (ET1610-43) at 1/500 dilution and competitor's antibody 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.

Fig2: Western blot analysis of Cytokeratin 5 on different lysates with Rabbit anti-Cytokeratin 5 antibody (ET1610-43) at 1/5,000 dilution.

Lane 1: A431 cell lysate
Lane 2: MDA-MB-468 cell lysate
Lane 3: MCF7 cell lysate (negative)
Lane 4: Mouse skin tissue lysate
Lane 5: Rat skin tissue lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 62 kDa
Observed band size: 57 kDa

Exposure time: 2 minutes 6 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 (ET1610-43) at 1/5,000 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.

	Fig3: Immunohistochemical analysis of paraffin-embedded human skin tissue with Rabbit anti-Cytokeratin 5 antibody (ET1610-43) at 1/800 dilution and competitor's antibody at 1/800 dilution. The section was not undergone antigen retrieval. 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 (ET1610-43) at 1/800 dilution and competitor's antibody at 1/800 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.
	Fig4: Immunohistochemical analysis of paraffin-embedded human Lung squamous cell carcinoma tissue with Rabbit anti-Cytokeratin 5 antibody (ET1610-43) at 1/1,000 dilution. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins. The section was incubated with ET1610-43 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
	Fig5: Immunohistochemical analysis of paraffin-embedded human skin tissue with Rabbit anti-Cytokeratin 5 antibody (ET1610-43) 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 (ET1610-43) 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.
	Fig6: Immunohistochemical analysis of paraffin-embedded human skin tissue with Rabbit anti-Cytokeratin 5 antibody (ET1610-43) at 1/1,500 dilution. The section was not undergone antigen retrieval. 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 (ET1610-43) at 1/1,500 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: Immunofluorescence analysis of paraffin-embedded human tonsil tissue labeling Cytokeratin 5 (ET1610-43) at 1/200 dilution and Vimentin (EM0401) 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 10% negative goat serum for 1 hour at room temperature, washed with PBS. And then probed with the primary antibodies Cytokeratin 5 (ET1610-43, red) at 1/200 dilution and Vimentin (EM0401, green) at 1/200 dilution at +4℃ overnight, washed with PBS. iFluorTM 594 conjugate-Goat anti-Rabbit IgG (HA1122) and iFluorTM 488 conjugate-Goat anti-Mouse IgG (HA1125)were used as the secondary antibodies at 1/1000 dilution. DAPI was used as nuclear counterstain.
	Fig8: Immunofluorescence analysis of paraffin-embedded human skin tissue labeling Cytokeratin 5 (ET1610-43) at 1/200 dilution and Vimentin (EM0401) 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 10% negative goat serum for 1 hour at room temperature, washed with PBS. And then probed with the primary antibodies Cytokeratin 5 (ET1610-43, red) at 1/200 dilution and Vimentin (EM0401, green) at 1/200 dilution at +4℃ overnight, washed with PBS. iFluorTM 594 conjugate-Goat anti-Rabbit IgG (HA1122) and iFluorTM 488 conjugate-Goat anti-Mouse IgG (HA1125)were used as the secondary antibodies at 1/1000 dilution. DAPI was used as nuclear counterstain.
	Fig9: Flow cytometric analysis of A431 cells labeling Cytokeratin 5. Cells were fixed and permeabilized. Then stained with the primary antibody (ET1610-43, 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).
	Fig10: Western blot analysis of Cytokeratin 5 on different lysates with Rabbit anti-Cytokeratin 5 antibody (ET1610-43) at 1/5,000 dilution. Lane 1: A431-si NT cell lysate Lane 2: A431-si Cytokeratin 5 cell lysate Lysates/proteins at 10 µg/Lane. Predicted band size: 62 kDa Observed band size: 57 kDa Exposure time: 5 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 (ET1610-43) at 1/5,000 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.

Fig11: Immunohistochemical analysis of paraffin-embedded mouse skin tissue with Rabbit anti-Cytokeratin 5 antibody (ET1610-43) at 1/1,000 dilution.

The section was not undergone antigen retrieval. 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 (ET1610-43) 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.

Fig12: Immunohistochemical analysis of paraffin-embedded rat skin tissue with Rabbit anti-Cytokeratin 5 antibody (ET1610-43) at 1/1,000 dilution.

The section was not undergone antigen retrieval. 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 (ET1610-43) 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.

Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.