Western blot analysis was carried out using standard methods. Total protein was extracted from the cultured cells or tumor tissues with lysis buffer. Protein concentration was determined by using a BCA method. The protein sample was then separated by 8-10% SDS-PAGE and transferred onto PVDF membranes. The membranes were incubated with primary antibodies overnight at 4°C. Membranes were probed with anti-RAB1B (1:1000), anti-Ras (1:5000), anti-Raf1 (1:1000), anti-Rac1 (1:500), anti-RhoA (1:5000), anti-CDC42 (1:1000), anti-β-actin (1:5000), and HRP conjugated anti-mouse (1:5000) and anti-rabbit (1:5000) (Abcam, Burlingame, CA).
Anti ras
Manufactured by Abcam
Sourced in United States
Anti-Ras is a lab equipment product that is used to detect and analyze the Ras protein, which is a key regulator of cell growth and differentiation. The product functions by binding to and identifying the Ras protein in biological samples, allowing researchers to study its expression and activity levels.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Abcam (3 mentions)
Anti e cadherin, by Abcam (2 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Anti rac1, by Abcam (1 mentions)
Anti cdc42, by Abcam (1 mentions)
Anti rhoa, by Abcam (1 mentions)
Anti raf1, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Anti cd9, by ABclonal (1 mentions)
Tanon 5200 chemiluminescent imaging system, by Shanghai Tanon Science & Technology (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
Anti tsg101, by ABclonal (1 mentions)
Anti cd63, by ABclonal (1 mentions)
Anti vimentin, by BD (1 mentions)
Anti tsg101, by Abcam (1 mentions)
Anti flag clone l5, by BioLegend (1 mentions)
Anti cpsf73, by Fortis Life Sciences (1 mentions)
9 protocols using anti ras
1
Western Blot Analysis of Signaling Proteins
2
Exosomal Protein Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins from exosomes and endothelial cells were collected and extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). The lysate was centrifuged at 12 000 rpm to obtain protein extracts. The protein concentration was measured using a bicinchoninic acid protein assay kit (Beyotime). Next, 15 μg of protein was loaded, separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes (Millipore, Mississauga, Canada). The membranes were incubated with the primary antibodies overnight at 4°C after being blocked with 5% nonfat milk for 1 h. Then, they were incubated with the appropriate secondary antibodies at room temperature for 1 h. The following primary antibodies were used: anti‐CD9, anti‐CD63, and anti‐TSG101 (1:1000; Abclonal, Woburn, MA, USA); anti‐SPRED1, anti‐Ras, and anti‐p/t‐Raf (1:1000; Abcam, Cambridge, UK); anti‐p/t‐MEK1/2 and anti‐p/t‐ERK1/2 (1:1000; Bioworld, Nanjing, China); and anti‐β‐actin (1:10000; Proteintech, Rosemont, IL, USA). The Tanon‐5200 Chemiluminescent Imaging System (Tanon Science & Technology, China) was used to detect the protein bands, after which the grayscale value was calculated using Image J software.
3
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins of cells or exosomes were extracted using radio immunoprecipitation assay (RIPA) lysis buffer, supplemented with a protease inhibitor, and quantified by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA). Western blot analysis was performed as previously described.46 (link) The following primary antibodies were used: anti-CD63 (cat. #ab217345; Abcam, Cambridge, UK), anti-TSG101 (cat. #ab125011; Abcam), anti-β-actin (cat. #ab179467; Abcam), anti-RasA1 (cat. #ab40677; Abcam), anti-Ras (cat. #ab52939; Abcam), anti-E-cadherin (cat. #610181; BD Biosciences), anti-vimentin (cat. #550513; BD Biosciences), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; cat. #ma5-15738; Thermo Fisher Scientific, Waltham, MA, USA).
4
Comprehensive Western Blot Antibody Panel
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti-hLin41 (R&D), anti-Lin28B (Cell Signaling Technology), anti-Lin28A (Abcam), anti-β-actin (clone C4, Millipore), anti-α-tubulin (clone TU-02, Santa Cruz Biotech), anti-CPSF73 (Bethyl Laboratories), anti-symplekin (BD Biosciences), anti-HuR (Santa Cruz Biotech), anti-Ras (Abcam), anti-eIF4GI [6 (link)], anti-FLAG (clone L5, BioLegend), and anti-HMGA2 (GeneTex) antibodies were used through the all western blot analysis.
5
Human NSCLC Cell Culture Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human NSCLC lines SK-LU-1, H441, H1437, Calu-6, and A549 were obtained from the American Type Culture Collection (Manassas, Virginia). SK-LU-1, H441, and H1437 cells were grown in Roswell Park Memorial Institute-1640 medium, Calu-6 cells were grown in Eagle 0 s minimal essential medium, and A549 cells were grown in Dulbecco 0 s modified Eagle medium/nutrient mixture F12; in all cases, the medium was supplemented with 10% fetal bovine serum. All cells were maintained at 37 C with a 5% CO 2 -containing humidified incubator. Antibodies against anti-CYP24A1, anti-RAS, and anti-b actin were purchased from Abcam (Cambridge, MA), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-AKT (T308) and total-AKT antibodies were obtained from Cell Signaling Technology (Waltham, MA).
6
Ras-GTP Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PVN samples were homogenized in a lysis buffer and Western blot was performed as previously described (Su Z. et al., 2014 (link)). Specifically, Ras active protein (Ras-GTP) was extracted from total tissue lysates by adding 80 ug GST-Raf1-RBD per 500 ug sample and using Active Ras Pull-Down and Detection Kit (Thermo scientific, USA) and assessed by western blot immediately or stored at −20°C.
Primary antibodies used included the following: anti-AT1-R (1:200, Millipore, USA), anti-p-ERK1/2 (1:300, Cell Signaling Technology, USA), anti-p-p38 (1:500, Cell Signaling Technology, USA), anti-p38 (1:1000, Cell Signaling Technology, USA), anti-Bax (1:500, Santa Cruz Biotechnology), anti-Bcl-2 (1:200, Santa Cruz Biotechnology), anti-Cleaved caspase-3 (1:600, Cell Signaling Technology), anti-ERK1/2 (1:1000, Cell Signaling Technology, USA), anti-Ras (1:200, Abcam Cambridge, UK), anti-β-actin (1:5000, Cell Signaling Technology), anti-GAPDH (1:1000, Cell Signaling Technology). The levels of the above proteins were determined as previously described (Su Z. et al., 2014 (link)). AT1-R protein content was normalized to GAPDH amounts. Ras, p-ERK1/2, and p-p38 levels were normalized to total Ras, ERK1/2 and p38, respectively. Bax, Bcl-2 and cleaved caspase-3 protein levels were normalized to β-actin.
Primary antibodies used included the following: anti-AT1-R (1:200, Millipore, USA), anti-p-ERK1/2 (1:300, Cell Signaling Technology, USA), anti-p-p38 (1:500, Cell Signaling Technology, USA), anti-p38 (1:1000, Cell Signaling Technology, USA), anti-Bax (1:500, Santa Cruz Biotechnology), anti-Bcl-2 (1:200, Santa Cruz Biotechnology), anti-Cleaved caspase-3 (1:600, Cell Signaling Technology), anti-ERK1/2 (1:1000, Cell Signaling Technology, USA), anti-Ras (1:200, Abcam Cambridge, UK), anti-β-actin (1:5000, Cell Signaling Technology), anti-GAPDH (1:1000, Cell Signaling Technology). The levels of the above proteins were determined as previously described (Su Z. et al., 2014 (link)). AT1-R protein content was normalized to GAPDH amounts. Ras, p-ERK1/2, and p-p38 levels were normalized to total Ras, ERK1/2 and p38, respectively. Bax, Bcl-2 and cleaved caspase-3 protein levels were normalized to β-actin.
7
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total protein was extracted from cell lysates and was separated by SDS‐PAGE. The protein was transferred to expanded polyvinylidene difluoride membranes (Pierce, Rockford, IL) with a Bio‐Rad Trans‐Blot system. After blocking, membranes were incubated with antibodies, including anti‐Ras (1:1000, Abcam, Cambridge, MA), anti–Janus kinase (JAK) (1:1000, Abcam), anti–phosphorylated JAK (1:1000, Abcam), anti‐PI3K (1:1000, Abcam), anti–phosphorylated PI3K (1:1000, Abcam), anti‐AKT (1:1000, Cell Signaling, Danvers, MA), anti–phosphorylated AKT (1:1000, Cell Signaling), anti‐Bcl‐2‐associated X protein (BAX) (1:1000, Cell Signaling), anti–cleaved caspase 3 (1:500, Cell Signaling), and anti–B‐cell lymphoma 2 (BCL‐2) (1:1000, Arigo Hsinchu, Taiwan, China), or anti‐GAPDH (1:5000, Sigma‐Aldrich Co, St. Louis, MO). Protein signals were obtained by ECL‐Western blotting system (AVEGENE CHEMX 400) and quantified by the ImageJ software (NIH, Bethesda, MD). GAPDH was used for the normalization of signal bands of other genes.
8
Protein Expression and Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
N6-Isopentenyladenosine (i6A ), 3-(N-Morpholino)propanesulfonic acid (MOPS) and NaCl-Sucrose were purchased from Sigma-Aldrich, Inc, St. Louis, MO, USA. The antibodies used: anti-Rap1A, anti-RAS, anti-caspase 3 (total and cleaved forms), anti- PARP (total and cleaved forms) were from Abcam, Cambridge, UK; anti-Caveolin-1 was from Santa Cruz Biotechnology, Dallas, TX, USA; anti-GAPDH and horseradish peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technologies, Danvers, MA, USA.
9
Molecular Techniques for Epigenetic Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All the chemicals used in this study were molecular biology grade as well as endotoxin free and used without further purification. Culture media, fetal bovine serum (FBS) and trypsin were purchased from Himedia and antibiotic from GIBCO. Trichostatin A (TSA), reagents for RNA isolation, cDNA synthesis Kit, Syber green for qRT-PCR, gene specific primer pairs were obtained from Sigma-Aldrich, St. Louis, MO, USA. Anti-DNMT1, anti-HDAC1, mouse monoclonal anti-β-actin and Goat anti-rabbit IgG-HRP antibodies, DNMT1 siRNA and scrambled (control) siRNA were purchased from Santa-Cruz Biotechnology. Anti-PARP (cleaved), anti-HDAC2, anti-E-cadherin, anti-N-cadherin, anti-vimentin, anti-α-tubulin, anti-γ-tubulin, anti-pericentrin, anti-TUBGCP2, anti-c-Myc, anti-Ras, anti-Cdk2 and anti-E2F1, anti-Bcl2 and anti-Bax antibodies were obtained from abcam.
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!