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Western blotAntibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [3]
- Immunocytochemistry [1]
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- Product number
- PA5-70104 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- K-Ras Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Reactivity
- Human, Rat
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 100 µL
- Concentration
- 0.5 mg/mL
- Storage
- -20° C, Avoid Freeze/Thaw Cycles
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis of K-Ras on rat lung tissue lysate. The sample was probed with a K-Ras polyclonal antibody (Product # PA5-70104) using a primary antibody dilution of 1.0 µg/mL.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Knockdown of KRAS was achieved by transfecting HCT 116 with K-Ras specific siRNAs (Silencer® select Product # s7939). Western blot analysis (Fig. a) was performed using whole cell extracts (1% SDS) from the KRAS knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with K-Ras Polyclonal Antibody (Product # PA5-70104, 1 µg/ml) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to KRAS.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot was performed using Anti-K-Ras Polyclonal Antibody (Product # PA5-70104) and a 22 kDa corresponding to KRAS was observed across cell lines and tissue extracts tested. The band around 25 kDa is probably due to ubiquitination of K-Ras. Whole cell extracts (30 µg lysate) of HCT116 (Lane 1), T-47D (Lane 2), SK-BR-3 (Lane 3), Caco-2 (Lane 4), HT-29 (Lane 5), SW480 (Lane 6), PANC-1 (Lane 7), NIH/3T3 (Lane 8) and tissue extracts of Mouse Brian (Lane 9), Mouse Lung (Lane 10) and Rat Brain (Lane 10) were electrophoresed using NuPAGE™ 12% Bis-Tris Protein Gel (Product # NP0342BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1 µg/ml) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescence analysis of K-Ras was performed using 70% confluent log phase HCT 116 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with K-Ras Rabbit Polyclonal Antibody (Product # PA5-70104) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415). Panel d represents the merged image showing Cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.
