RPMI-8226 cells (obtained from ATCC) were incubated with test compounds for 48 hrs. Whole cell lysate was obtained using RIPA buffer (0.15 M NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% Triton (v/v) X-100, 0.05 M Tris HCl) containing protease and phosphatase inhibitors. Protein content was determined using the bicinchoninic acid (BCA) method (Pierce Chemical). Equivalent amounts of cell lysate were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membrane, probed with the appropriate primary antibodies (anti-H-Ras (610001, BD Transduction Laboratories), anti-Rap1a (sc-373968, Santa Cruz Biotechnology), anti-β-tubulin (T-5201, Sigma)), and detected using HRP-linked secondary antibodies and Clarity ECL (for tubulin) or Millipore Immobilon ECL (for Ras and Rap1a) western blotting reagents per manufacturer’s protocols.
Anti h ras
Manufactured by BD
Sourced in United States
Anti-H-Ras is a laboratory reagent used for the detection and quantification of the H-Ras protein. It functions as an antibody that specifically binds to the H-Ras protein, enabling its identification and measurement in various experimental and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti β tubulin, by Merck Group (2 mentions)
Bca method, by Thermo Fisher Scientific (1 mentions)
Imagequant las 4000 imaging system, by GE Healthcare (1 mentions)
Anti akt and anti phospho akt s473, by Cell Signaling Technology (1 mentions)
Ecl kit, by GE Healthcare (1 mentions)
Anti htert, by Abcam (1 mentions)
Anti rap1a, by Santa Cruz Biotechnology (1 mentions)
Millipore immobilon ecl, by Merck Group (1 mentions)
Bicinchoninic acid bca method, by Thermo Fisher Scientific (1 mentions)
3 protocols using anti h ras
1
Western Blot Analysis of Ras and Rap1a
2
Immunoblot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed twice with cold PBS and lysed in a buffer containing 0.5% NP-40, 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 1 mM PMSF, and 1 mM Na3VO4. The lysate was clarified by centrifugation at 15,000 rpm for 10 min, and the supernatants were subjected to SDS-PAGE in 10–12% polyacrylamide gels. Separated proteins were transferred to polyvinylidene difluoride membranes and blocked with Tris-buffered saline (TBS) containing Tween-20 (TBS-T) and 5% skim-milk at room temperature for 1 h. The membranes were incubated with primary antibodies at 4°C overnight, washed with TBS-T, incubated with secondary antibodies, and washed three more times with TBS-T. Immunoreactive signals were then detected by using an enhanced chemiluminescence (ECL) kit (GE Healthcare, Buckinghamshire, UK) and an ImageQuant LAS4000 imaging system (GE Healthcare). Primary antibodies were as follows: anti-hTERT (Abcam, Cambridge, UK); anti-Akt and anti-phospho-Akt (S473) (Cell Signaling Technology, Beverly, MA, USA); anti-SV40 T-antigen (clone Ab-1 for large T-antigen and clone Ab-3 for small T-antigen) (Oncogene Research Products, Dublin, OH, USA); and anti-H-Ras (BD Transduction Laboratories, Lexington, KY, USA).
3
Evaluating Triazole Inhibitors in RPMI-8226 Cells
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!