- 415700 - Invitrogen Antibodies KRAS antibody

Submitted references A Novel KRAS Antibody Highlights a Regulation Mechanism of Post-Translational Modifications of KRAS during Tumorigenesis.

Assi M, Pirlot B, Stroobant V, Thissen JP, Jacquemin P
International journal of molecular sciences 2020 Sep 2;21(17)

Integrated analysis of patient samples identifies biomarkers for venetoclax efficacy and combination strategies in acute myeloid leukemia.

Zhang H, Nakauchi Y, Köhnke T, Stafford M, Bottomly D, Thomas R, Wilmot B, McWeeney SK, Majeti R, Tyner JW
Nature cancer 2020 Aug;1(8):826-839

CRISPR screens in cancer spheroids identify 3D growth-specific vulnerabilities.

Han K, Pierce SE, Li A, Spees K, Anderson GR, Seoane JA, Lo YH, Dubreuil M, Olivas M, Kamber RA, Wainberg M, Kostyrko K, Kelly MR, Yousefi M, Simpkins SW, Yao D, Lee K, Kuo CJ, Jackson PK, Sweet-Cordero A, Kundaje A, Gentles AJ, Curtis C, Winslow MM, Bassik MC
Nature 2020 Apr;580(7801):136-141

Targeting wild-type KRAS-amplified gastroesophageal cancer through combined MEK and SHP2 inhibition.

Wong GS, Zhou J, Liu JB, Wu Z, Xu X, Li T, Xu D, Schumacher SE, Puschhof J, McFarland J, Zou C, Dulak A, Henderson L, Xu P, O'Day E, Rendak R, Liao WL, Cecchi F, Hembrough T, Schwartz S, Szeto C, Rustgi AK, Wong KK, Diehl JA, Jensen K, Graziano F, Ruzzo A, Fereshetian S, Mertins P, Carr SA, Beroukhim R, Nakamura K, Oki E, Watanabe M, Baba H, Imamura Y, Catenacci D, Bass AJ
Nature medicine 2018 Jul;24(7):968-977

The proto-oncogene KRAS is targeted by miR-200c.

Kopp F, Wagner E, Roidl A
Oncotarget 2014 Jan 15;5(1):185-95

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on tissue and membrane enriched extracts (30 µg lysate) of HCT-116 (Lane 1), K562 (Lane 2) and Jurkat (Lane 3). The blots were probed with Anti-K-Ras Mouse Monoclonal Antibody (Product # 415700, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP conjugate (Product # 62-6520, 1:4000 dilution). A ~ 21 kDa band corresponding to K-Ras was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12% Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on tissue and membrane enriched extracts (30 µg lysate) of HCT-116 (Lane 1), K562 (Lane 2) and Jurkat (Lane 3). The blots were probed with Anti-K-Ras Mouse Monoclonal Antibody (Product # 415700, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP conjugate (Product # 62-6520, 1:4000 dilution). A ~ 21 kDa band corresponding to K-Ras was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12% Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of K-Ras was done on 70% confluent log phase MCF-7 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with K-Ras (9.13) Mouse Monoclonal Antibody (Product # 415700) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing cytoplasmic localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of KRAS showing staining in the cytoplasm of paraffin-embedded human colon carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10 mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with KRAS monoclonal antibody (Product # 415700) diluted in 3% BSA-PBS at a dilution of 1:50 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using a HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of KRAS showing staining in the cytoplasm and membrane of paraffin-embedded human prostate carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10 mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with KRAS monoclonal antibody (Product # 415700) diluted in 3% BSA-PBS at a dilution of 1:50 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using a HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 KRAS is a predicted target of miR-200c and its protein expression inversely correlates with miR-200c expression in breast cancer cells A) Target site prediction of miR-200c in the 3'UTR of the KRAS gene. By means of the three different prediction algorithms TargetScan, miRanda and DIANA microT, a unique conserved binding site with a 7mer-m8 seed at position 305 - 311 of the 3'UTR of the KRAS gene was found. B) miR-200c expression in a panel of breast cancer cell lines. miR-200c expression was normalized to miR-191 and values are stated as mean +- SD (n=3). C) K-ras protein expression in a panel of breast cancer cell lines. Total cell lysates were subjected to Western blot analysis and incubated with indicated antibodies. D) Correlation of K-ras protein and miR-200c expression. The values of the relative K-ras protein and miR-200c expression are listed in Table 1 . The graph shows the Pearson correlation scatter plot of the relative K-ras and miR-200c levels in the different breast cancer cell lines (*p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2 miR-200c inhibits K-ras protein expression without affecting KRAS mRNA levels A) Luciferase reporter assay with different breast cancer cell lines. The renilla luciferase reporter containing the 3'UTR of KRAS including the predicted target site of miR-200c (RLuc) or the firefy luciferase control plasmid pGL3 (FLuc) were transfected into the indicated cell lines. Renilla reporter luciferase activity was normalized to the activity of the firefy control as ratio (RLuc/FLuc). Values are stated as mean +- SEM (n=5). B) Luciferase reporter assay upon miR-200c modulation. MDA-MB-436 cells were transfected with either pre-miR-200c (pre) or scrambled pre-miR-control (ctr). BT-474 cells were transfected with either miR-200c inhibitor (inh) or scrambled control inhibitor (ctr). Relative luciferase activities (RLuc/FLuc) are depicted in the graph. Values are stated as mean +- SEM (n=5). For statistical analysis a student's t-test was performed (**p

415700

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