CALU-6 cells were seeded in growth media at 200 cells per chamber in Permanox 4-chamber slides (Fisher Scientific, 1256521). The following day, media was replaced with treatment media containing TVB-3166 and cells were incubated for 95.5 h. Fluorescent staining of lipid raft domains was performed using cholera toxin patching method as described (Janes et al., 1999 (link), Abulrob et al., 2004 (link)). Fixation was performed in 4% formaldehyde for 20 min on ice. Permeabilization was performed in PBS with 0.1% Triton X-100 for 10 min at room temperature. Blocking was performed in PBS with 1% BSA for 30 min at room temperature. N-Ras antibody (Santa Cruz Biotechnology, sc-519) diluted 1:250 in PBS with 0.1% BSA was added and incubated overnight at 4 °C. Anti-rabbit Alexa-fluor 594 antibody (Life Technologies, ab150076) diluted 1:500 in PBS with 0.1% BSA was added and incubated for 1 h at room temperature. After 4 × PBS washes, slides were mounted with coverslips using Prolong Gold Antifade mounting medium with DAPI (Life Technologies, P36935) and hardened overnight. Imaging was performed using a Zeiss LSM510 confocal microscope.
N ras antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The N-RAS antibody is a tool used for the detection and analysis of the N-RAS protein, a member of the RAS family of small GTPases. It can be used in various laboratory techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry, to study the expression, localization, and interactions of the N-RAS protein in biological samples.
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Lab products found in correlation
Prolong gold antifade mounting medium with dapi, by Thermo Fisher Scientific (1 mentions)
Lsm 510 confocal microscope, by Zeiss (1 mentions)
Desmin, by Cell Signaling Technology (1 mentions)
Caspase 9, by Cell Signaling Technology (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Ecl detection kit, by GE Healthcare (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
U2af65, by Santa Cruz Biotechnology (1 mentions)
Fgf2 antibody, by Santa Cruz Biotechnology (1 mentions)
Ecl system, by Cytiva (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
5 protocols using n ras antibody
1
Imaging Lipid Rafts in CALU-6 Cells
2
Protein Expression Analysis in Cells
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Rabbit antibodies against PKD1, PKD2, PKD3, Ki67, caspase9, and desmin were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti‐rabbit secondary antibody, anti‐mouse secondary antibody, and β‐actin antibody were purchased from Kangchen, anti‐ELAVL1 antibody was purchased from Absci. N‐Ras antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Western blot was carried out following the standard procedure. Briefly, protein lysates were separated by SDS‐PAGE, transferred to PVDF membranes, and immunoblotted with the respective antibodies as indicated above and in the figures. Blots were developed with SuperSignal West Femto Maximum Sensitivity Substrate (Pierce/Thermo Scientific, Rockford, IL, USA).
3
Detecting Small GTPases in Cells
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N-, K-, and H-RAS were detected with a 1:100 dilution of murine monoclonal paN-RAS antibody (BD Biosciences, San Diego, CA, USA, product #610002). N-RAS was detected with a 1:400 dilution of a rabbit polyclonal N-RAS antibody (Santa Cruz, CA, USA, product #sc-519). RAB5A was detected with a 1:250 dilution of a rabbit polyclonal RAB5A antibody (Santa Cruz, Santa Cruz, CA, USA, product #sc-309). Non-prenylated RAP1A was detected with a 1:250 dilution of a goat polyclonal RAP1A antibody (Santa Cruz, product #1482). GAPDH was detected with a 1:1000 dilution of rabbit polyclonal antibody to GAPDH (Cell Signaling, Boston, MA, USA, product #2118). β-actin was detected with a 1:25,000 dilution of murine monoclonal antibody to β-actin (Sigma, St. Louis, MI, USA, product #A5441). The horseradish peroxidase-conjugated secondary antibodies used were goat anti-rabbit (Pierce Biotechnology, Rockford, IL, USA, product #1858415), donkey anti-rabbit (GE Healthcare, Piscataway, NJ, USA, product #NA934V), or sheep anti-mouse (GE Healthcare, product number #NA931V). Immune complexes were visualized with an ECL detection kit (GE Healthcare, Piscataway, NJ, USA) and recorded on X-ray film.
4
Protein Extraction and Immunoblotting Procedure
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Cells were lysed with a lysis buffer (20 mM Tris [pH 7.5], 0.1% Triton X-100, 0.5% deoxycholate, 1 mM PMSF, 10 μg/ml aprotinin, and 10 μg/ml leupeptin) and then cleared via centrifugation at 4°C. The total protein concentration was determined using a Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions, and the obtained proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After transferring the proteins onto nitrocellulose membranes and blocking the membranes, the membranes were incubated overnight at 4°C with antibodies in phosphate-buffered saline containing 0.1% Tween 20. The primary antibodies employed for immunoblotting were as follows: anti-β-actin antibody (Santa Cruz Biotechnology), anti-KRT1 antibody (Abcam), anti-KRT10 antibody (Covance, Richmond, VA, USA), anti-p53 antibody (Wako), p44/42 ERK antibody (Cell Signaling Inc., Beverly, MA, USA), phospho-p44/42 ERK antibody (Cell Signaling Inc.), U170K antibody (Santa Cruz Biotechnology), U2AF65 (Santa Cruz Biotechnology), PRP8 antibody (Santa Cruz Biotechnology), FGF2 antibody (Santa Cruz Biotechnology), and NRAS antibody (Santa Cruz Biotechnology). All protein bands were detected using an ECL system (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
5
Immunohistochemical Analysis of N-RAS
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A total of 43 paraffin-embedded tumor specimens were selected for this study. N-RAS antibody (1:50, Santa Cruz Biotechnology, Dallas, TX, USA) was the primary antibody used, and phosphate buffered saline (PBS) was used as a negative control. All immunostained sections were blindly independently evaluated by two pathologists. The intensity of immunostaining (negative =0, light yellow =1, light brown =2, brown =3) and the percentage of positive tumor cells (≤5%=0, >5%–25%=1, >25%–50%=2, >50%–75%=3, >75%=4) were assessed in at least five high-power fields (400× magnification). The final scores were multiplied by the intensity score and percentage score.
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