Total protein from tissues and cells was extracted according to the manufacturer’s recommended protocol (Vazyme, Nanjing, China). Protein concentrations were determined using a BCA Protein Assay Kit (Vazyme, Nanjing, China). Samples with equal amounts of protein (50 μg) were fractionated on 10% SDS-polyacrylamide gels, transferred to polyvinylidene difluoride membranes and blocked in 5% skim milk in TBST for 1.5 h at 25 ± 1 °C. The membranes were then incubated at 4 °C overnight with 1:1000 dilutions (v/v) of the primary antibodies (anti-Lin28B (#ab191881, Abcam, Shanghai, China) and anti-NRAS (#ab154291, Abcam, Shanghai, China)). After the membranes were washed with TBST, incubations with 1:4000 dilutions (v/v) of the secondary antibodies were conducted for 2 h at 25 ± 1 °C. Protein expression was detected using an Enhanced Chemiluminescence Detection System (ImageQuant LAS 4000 mini, USA). β‐Actin was used as a loading control.
Ab154291
Manufactured by Abcam
Sourced in China
Ab154291 is a laboratory reagent designed for use in research applications. It is a purified antibody that can be used to detect a specific target molecule or protein. The core function of this product is to provide researchers with a tool for identifying and quantifying the presence of the target in their samples.
Automatically generated - may contain errors
Lab products found in correlation
Erk1 2, by Cell Signaling Technology (2 mentions)
Phospho mek1 2, by Cell Signaling Technology (2 mentions)
Phospho erk1 2, by Cell Signaling Technology (2 mentions)
Mek1 2, by Cell Signaling Technology (2 mentions)
Ab8245, by Abcam (2 mentions)
Ab191881, by Abcam (1 mentions)
Bca protein assay kit, by Vazyme (1 mentions)
Polyvinylidene fluoride pvdf membrane, by Merck Group (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Radio immunoprecipitation assay ripa buffer, by Beyotime (1 mentions)
Bicinchoninic acid kit, by Beyotime (1 mentions)
Ecl kit, by Cytiva (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab178886, by Abcam (1 mentions)
Ab214362, by Abcam (1 mentions)
Ab17942, by Abcam (1 mentions)
Ripa kit, by Beyotime (1 mentions)
Ab9485, by Abcam (1 mentions)
Ab131004, by Abcam (1 mentions)
5 protocols using ab154291
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of NRAS, MEK, and ERK
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot was performed as we previously described [17 (link)]. Primary antibodies used were as follows: for NRAS, ab154291, 1:1000, Abcam; for GAPDH, ab8245, 1:5000, Abcam; for phospho-MEK1/2, #9154, 1:1000, Cell Signaling Technology; for MEK1/2, #8727, 1:1000, Cell Signaling Technology; for phospho-ERK1/2, #4370, 1:2000, Cell Signaling Technology; for ERK1/2, #4695, 1:1000, Cell Signaling Technology.
3
Western Blot Analysis of NRAS Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RB cells were lysed with the Radioimmunoprecipitation Assay (RIPA) buffer (Beyotime, Shanghai, China), and protein samples were prepared. Protein concentration was measured using a bicinchoninic acid kit (Beyotime, Shanghai, China). Fifteen microliters of protein samples from each group were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to Polyvinylidene Fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After blocking with 5% skimmed milk, the membrane was incubated with a primary antibody (anti-NRAS antibody, rabbit anti-human polyclonal antibody, 1:1000, ab154291; Abcam, Shanghai, China) at 4°C overnight and washed with Tris-buffered saline with Tween 20 (TBST, Beyond, Shangai, China). After that, the membrane was incubated with the secondary antibody (goat anti-rabbit IgG, 1:2000, ab205718; Abcam, Shanghai, China) at 25°C for 2h. Protein bands were developed using an ECL kit (Amersham Pharmacia Biotech, Little Chalfont, UK), and the images were captured using an ImageQuant LAS4000 miniature biomolecular imager (GE Healthcare, Chicago, IL, USA). GAPDH was used as an internal reference. ImageJ (v.1.8.0 NIH, Bethesda, MD, USA) was used for the quantitative analysis of the gray values for the protein bands.
4
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from samples using RIPA Kit (Beyotime Biotechnology, China). The protein concentration was quantified using Bradford method. Proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and thereafter transferred to PVDF membrane which was blocked in Tris‐Buffered Saline Tween (TBST) containing 0.1%Tween‐20 and 5% low‐fat milk for 1 h. The primary antibody was added and incubated overnight at 4°C and then the secondary antibodies were added and incubated at room temperature for 1 h. Lastly, the ECL solution was applied to the membranes, and the membranes together with a film were placed in a visualizer for further analysis. The ImageJ software was used to measure the intensities. Primary antibodies for NRAS (ab154291), BRAF (ab151286), MEK1/2 (ab178886), pMEK1/2 (phospho s218, s222 and s226, ab78132), ERK1/2 (ab17942), pERK1/2 (phospho thr202 and thr204, ab214362), and GAPDH (ab9485) were all purchased from Abcam, whereas antibodies for PI3K‐p110α (#4249), PI3K‐p110β (#3011s), AKT (#9272), pAKT‐ser473 (#4060), pAKT‐thr308 (#2965), PTEN (#4005), Cyclin D1 (#2926), and p27 (#3686) were purchased from Cell Signaling. HRP labeled goat anti‐mouse and goat anti‐rabbit as secondary antibodies were purchased from Beyotime Biotechnology.
5
Western Blot Antibody Optimization Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot was conducted as previously described (38 (link)) with the primary antibodies: for NF90, ab131004, 1:2,000, Abcam; for EZH2, #07-689, 1:2,000, Millipore; for NRAS, ab154291, 1:1,000, Abcam; for GAPDH, ab8245, 1:5,000, Abcam; for phospho-MEK1/2, #9154, 1:1,000, Cell Signaling Technology; for MEK1/2, #8727, 1:1,000, Cell Signaling Technology; for phospho-ERK1/2, #4370, 1:2,000, Cell Signaling Technology; for ERK1/2, #4695, 1:1,000, Cell Signaling Technology.
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!