Protein lysates from vehicle‐ and Zarnestra‐treated Panc‐1 and BxPC‐3 cells were incubated with a Raf‐1 pulldown reagent linked to agarose beads as per manufacturer's instructions (Cat no. 16117, EMDMillipore, Etobicoke, ON, Canada). Lysates were then separated on an SDS/PAGE and immunoblotted using a RAS antibody (Cat no. 05‐516 Clone RAS10, EMDMillipore).
Ras10
Manufactured by Merck Group
Sourced in Morocco
The RAS10 is a laboratory equipment product manufactured by Merck Group. It is designed for specific laboratory applications, but a detailed description cannot be provided while maintaining an unbiased and factual approach. More information may be available from the product's technical documentation or from Merck Group directly.
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Lab products found in correlation
P paxillin, by Cell Signaling Technology (2 mentions)
Gapdh, by SCBT (2 mentions)
P pak1, by Cell Signaling Technology (2 mentions)
Cathepsin b, by SCBT (2 mentions)
P erk, by SCBT (2 mentions)
P jnk, by SCBT (2 mentions)
Vinculin, by SCBT (2 mentions)
α actinin, by SCBT (2 mentions)
Talin, by SCBT (2 mentions)
P pi3k, by SCBT (2 mentions)
Rac 1, by SCBT (2 mentions)
Mekk 1, by SCBT (2 mentions)
Laminin, by SCBT (2 mentions)
Protein a g micro beads, by Miltenyi Biotec (2 mentions)
Ecl solution, by Cytiva (1 mentions)
Ha 11, by Fortrea (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Hrp conjugated anti mouse secondary antibody, by Agilent Technologies (1 mentions)
Ras activation assay kit, by Merck Group (1 mentions)
Pcdna3.1 expression vector, by Thermo Fisher Scientific (1 mentions)
7 protocols using ras10
1
Raf-1 Pulldown Assay for Ras Detection
2
Immunoblotting for Protein Expression
3
Protein Expression Analysis by Western Blot
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Total protein (40 μg) was separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was then blocked with 5% non-fat milk for 1 h, incubated with primary antibody overnight at 4 °C, washed thrice with PBS-T (PBS plus 0.1% Tween 20), and incubated with HRP-linked secondary antibody for 1 h at room temperature. The membrane was then washed and bands were visualized by chemiluminescence assay. The following antibodies were used: uPAR, cathepsin B, ERK, p-ERK, JNK, p-JNK, p38, p-p38, Vinculin, α-Actinin, Talin, PI3K, p-PI3K, Rac-1, MEKK-1, Laminin and GAPDH (all from SCBT, Santa Cruz, CA). We also used antibodies for Paxillin, p-Paxillin, Pak-1 and p-Pak-1 (all from Cell Signaling Technology, Danvers, MA). We obtained Ras10 from Millipore (Billerica, MA).
For immunoprecipitation, cell lysates (300 μg) were pre-cleared by protein A/G micro-beads (Miltenyi Biotec, Auburn, CA) and then incubated with specific antibodies at a dilution of 1:100 overnight at 4 °C. The beads were washed with lysis buffer and resuspended in sample buffer before the immunoprecipitated protein was subjected to immunoblotting.
For immunoprecipitation, cell lysates (300 μg) were pre-cleared by protein A/G micro-beads (Miltenyi Biotec, Auburn, CA) and then incubated with specific antibodies at a dilution of 1:100 overnight at 4 °C. The beads were washed with lysis buffer and resuspended in sample buffer before the immunoprecipitated protein was subjected to immunoblotting.
4
KRAS Mutagenesis and RAS Activation Assay
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Full-length of KRAS cDNA was cut from pBabe K-Ras 12V vector (Addgene plasmid 12544)37 (link) and cloned into pcDNA3.1 (+) expression vector (Invitrogen) via BamH1 and Xba1 restriction sites. Corresponding KRAS mutations were introduced into the expression vector using QuickChange® II Site-Directed Mutagenesis Kit according to the manufacturer’s recommendations (Stratagene). The desired mutations in each construct were finally confirmed by direct sequencing. The primer sequences for mutagenesis were listed in Table 5 . Ras Activation Assay Kit (Millipore) was used to measure the level of active RAS (RAS-GTP) after transient transfection of corresponding plasmid into the cell lines. In brief, 0.5 mg of cell extract was immunoprecipitated with agarose beads containing human Ras Binding Domain (RBD, residues 1–149) of Raf-1. After washing, the beads were mixed with protein loading buffer and 10% of the mixture was electrophoresed by 12% SDS-PAGE for western blot analysis as previously described.38 (link),39 (link) The primary antibodies used were pan-RAS (RAS10, Millipore; 1:2000) and p-ERK1/2 (9102, Cell Signaling; 1:1000). HRP conjugated anti-mouse secondary antibody used was purchased from DAKO (1:20000 dilution).
5
Protein Expression Analysis by Western Blot
6
Ras Protein Immunoprecipitation and Detection
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The anti-DA-Raf pAb was coupled to Protein A Sepharose 4 Fast Flow (GE Healthcare). RLE cells were lysed with the lysis buffer, and the lysates were incubated with the antibody-coupled Sepharose for 60 min at 4°C [50 (link)]. After thorough washing with the lysis buffer, bound proteins were dissociated with the SDS sample buffer and detected by immunoblotting with anti-Ras mouse mAb RAS10 (Millipore).
7
Quantifying Ras-GTP Activation
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