Proteins were extracted from cells with a lysis buffer containing 50 mM Tris (pH 7.5), 5 mM EDTA, 150 mM NaCl, 0.25% Triton X-100 and 1 mM DTT. Protease inhibitors (Complete protease inhibitor cocktail, Roche) and phosphatase inhibitors (50 mM NaF, 1 mM Na3VO4) were added to the basic buffer. Twenty microgram proteins were electrophoresed on a SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane. After a one-hour blocking in Tris buffered saline Tween 0.1% (TBST) containing 5% milk, membranes were incubated overnight at 4 °C with mouse monoclonal RhoA (Santa Cruz, sc-418, 1:1000), rabbit polyclonal RhoB (Santa Cruz, sc-180, 1:1000) and tubulin (Santa Cruz, 1:500) primary antibodies. Membranes were then washed three times in TBST and incubated for one hour with horseradish peroxidase-conjugated secondary antibody (1:2000). Detection was then performed with the ECL detection system (GE Healthcare).
Sc 418
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-418 is a lab equipment product offered by Santa Cruz Biotechnology. It is designed for use in research and laboratory settings. The core function of Sc-418 is to facilitate specific tasks or experiments, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Szh stereo zoom microscope, by Olympus (2 mentions)
Novolink polymer detection system, by Leica Biosystems (2 mentions)
Af835, by R&D Systems (2 mentions)
Active rhoa detection kit, by Cell Signaling Technology (2 mentions)
Sc 126, by Santa Cruz Biotechnology (2 mentions)
Sc 7974, by Santa Cruz Biotechnology (2 mentions)
Sc 81405, by Santa Cruz Biotechnology (2 mentions)
Sc 81406, by Santa Cruz Biotechnology (2 mentions)
Sc 180, by Santa Cruz Biotechnology (1 mentions)
Ecl detection system, by GE Healthcare (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
P47phox, by Santa Cruz Biotechnology (1 mentions)
Nupage 4 12 bis tris precast gel, by Thermo Fisher Scientific (1 mentions)
C3956, by Abcam (1 mentions)
Las 4000, by Fujifilm (1 mentions)
Ab108347, by Abcam (1 mentions)
Ab77581, by Abcam (1 mentions)
Ab56510, by Abcam (1 mentions)
Ab2899, by Abcam (1 mentions)
Cl xposure film, by Thermo Fisher Scientific (1 mentions)
25 protocols using sc 418
1
Western Blot Analysis of Rho GTPases
2
Fractional Subcellular Extraction and Immunoblotting of Rat MAB
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The rat MAB was homogenized in lysis buffer containing 50 mmol/L Tris-HCl (pH
7.4), 2.5 mmol/L EDTA, 5 mmol/L EGTA, and protease inhibitor. Homogenates were
centrifuged at 100,000 × g for 1 h, at 4ºC. The supernatant (cytosolic
fraction) was collected. The pellet, containing the particulate fraction, was
resuspended in lysis buffer containing 1% Triton X-100 and centrifuged at 10,000
× g for 10 min at 4 °C. Membranes were then incubated with specific
antibodies for RhoA (diluted 1:1000, sc-418, Santa Cruz Biotechnology, Dallas,
TX, USA) or p47phox (diluted 1:500, Santa Cruz Biotechnology) as previously
published.21 (link)
7.4), 2.5 mmol/L EDTA, 5 mmol/L EGTA, and protease inhibitor. Homogenates were
centrifuged at 100,000 × g for 1 h, at 4ºC. The supernatant (cytosolic
fraction) was collected. The pellet, containing the particulate fraction, was
resuspended in lysis buffer containing 1% Triton X-100 and centrifuged at 10,000
× g for 10 min at 4 °C. Membranes were then incubated with specific
antibodies for RhoA (diluted 1:1000, sc-418, Santa Cruz Biotechnology, Dallas,
TX, USA) or p47phox (diluted 1:500, Santa Cruz Biotechnology) as previously
published.21 (link)
3
Western Blot Analysis of Protein Expressions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extracts were separated on NuPAGE 4–12% Bis-Tris pre-cast gels (Invitrogen) for 1 h at 200 V and transferred onto a methanol-activated PVDF membrane at 400 mA for 1 h. The following primary antibodies were used: anti-Myc (rabbit polyclonal, 1/4000, Sigma-Aldrich C3956), anti-Otx2 (rabbit monoclonal, 1/2000, Abcam ab92326), anti-RhoA (mouse monoclonal, 1/200, Santa Cruz sc418). Membranes were imaged with a LAS-4000 gel imager (Fujifilm) and quantified by densitometry with ImageJ.
4
Western Blot Validation of Knockdown Experiments
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For verification of knockdown experiments, cells were lysed in RIPA buffer 48 h after transfection. Extracted proteins were separated by 4–20% SDS-PAGE (Thermo Fisher Scientific), transferred to PVDF membranes (Bio-Rad Laboratories), incubated at 75 V for 2 h, and blocked for 1 h with 5% nonfat milk (Bio-Rad Laboratories) or BSA (Sigma-Aldrich). The PVDF membranes were incubated overnight at 4°C with appropriate antibodies: anti-ARF1 (dilution 1:1,000; ab108347; Abcam); anti-ARF6 (dilution 1:1,000; ab77581; Abcam); anti-ARNO (dilution 1:1,000; ab56510; Abcam); anti–cytohesin 1 (dilution 1:500; MABT14; EMD Millipore); anti–α-tubulin (dilution 1:3,000; T6199; Sigma-Aldrich); anti-βCOP (dilution 1:1,000; ab2899; Abcam); anti-HA (dilution 1:1,000; 2367; Cell Signaling Technology); anti-Cdc42 (dilution 1:1,000; 2462; Cell Signaling Technology); anti-RhoA (dilution 1:1,000; sc-418; Santa Cruz Biotechnology, Inc.); anti-Rac1 (dilution 1:1,000; 610650; BD); and anti–NM myosin-IIA (dilution 1:1,000; M8064; Sigma-Aldrich).
After three washes (10 min each), appropriate secondary antibodies conjugated with HRP (Bio-Rad Laboratories) were incubated with the membrane for 1 h, washed three times (15 min at RT), and detected by ECL Western blotting substratum (Thermo Fisher Scientific) using CL-Xposure film (Thermo Fisher Scientific).
After three washes (10 min each), appropriate secondary antibodies conjugated with HRP (Bio-Rad Laboratories) were incubated with the membrane for 1 h, washed three times (15 min at RT), and detected by ECL Western blotting substratum (Thermo Fisher Scientific) using CL-Xposure film (Thermo Fisher Scientific).
5
Western Blot Analysis of RhoA and Actin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analyses were performed as described previously [7 (link)]. Briefly, complete lysate proteins were separated using 15% SDS-PAGE and subsequently transferred onto nitrocellulose membranes (GE Healthcare UK Limited, Buckinghamshire, UK) and blocked with 5% (w/v) non-fat dried milk (Sucofin TSI GmbH, Zeven, Germany). For Western blot analysis the following primary antibodies were used: RhoA was identified using a mouse monoclonal IgG from Santa Cruz Biotechnologies sc-418 (Santa Cruz, CA, USA) and Actin (Sigma-Aldrich Chemie GmbH, Munich, Germany) was used as loading control. For the chemiluminescence reaction, ECL Femto (Pierce, Thermo Fisher Scientific Inc., Rockford, IL, USA) was used. All signals were analyzed densitometrically using the KODAK 1D software (Version 3.5, KODAK GmbH, Stuttgart, Germany, 2001) and normalized to β-Actin signals.
6
RhoA GTPase Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The suspension and attached osteoclasts from adhesion assay were lysed in a buffer containing 50 mM Tris/HCl, pH 7.5, 1 mM EDTA, 500 mM NaCl, 10 mM MgCl2, 1% Triton X-100, and protease inhibitors (4 µg/ml leupeptin, 5 µg/ml apropeptin, 1 mM PMSF, 5 mM NaF, 5 mM sodium orthovanadate and protease inhibitor cocktail). Cell lysates were clarified by centrifugation at 15 000 g at 4 °C for 10 min. Equal volumes of lysates were incubated with 50 µg GST–RBD fusion protein at 4 °C for 1 hr while rotating. After pulldown, the beads were centrifuged at 250 g for 2 min and 4 °C and then washed five times with a buffer containing 50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml apropeptin and 1 mM PMSF. The pulled down active RhoA proteins were analyzed by SDS-PAGE followed by immunoblotting against RhoA (Santa Cruz Biotechnology, sc-418). ImageJ was used to quantify the immunoblot band density that reflects the amount of proteins, and the relative amount of GTP-bound RhoA was normalized to the total amount of RhoA in cell lysates for the comparison of RhoA activity in different samples.
7
Analyzing Plac8 Protein Localization
8
Immunohistochemical Profiling of Metastatic LUAD
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IHC assays were performed to detect the expression of ALPL, p-ERK, c-Myc and RhoA in formalin-fixed paraffin-embedded metastatic lung tumor tissues of mice injected with LUAD cells. Specific antibodies against ALPL (GTTX100817, Genetex), p-ERK1/2 (4370S, CST), c-Myc (sc-764, Santa Cruz) and RhoA (sc-418, Santa Cruz) were used for IHC staining, which were performed using a kit from Boster Bio-Engineering Company (SA1022; Wuhan, China). Immunostained images were captured with the Nikon Eclipse Ni microsystem (DS-Ri2) and analyzed with Image-Pro Plus version 6.0 (Media Cybernetics, Rockville, MD, USA) by calculating the integrated optical density (IOD) of each stained area (IOD/area). At least five images per specimen were counted.
9
Intestinal Tumor Characterization in Mice
10
Investigating Rho Signaling in Axons
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!