Control empty vector or FIRRE overexpressed cells were lysed in a sample buffer, subjected to SDS-PAGE, and transferred onto a nitrocellulose membrane. The membrane was blocked in 5% nonfat dried milk and incubated with a specific primary antibody overnight at 4 °C followed by incubation with a secondary antibody conjugated with horseradish peroxidase (HRP) for 1 h. Proteins were detected by an enhanced chemiluminescence Western blot substrate (ThermoFisher Scientific). Commercially available antibodies to cyclin D1 (Santa Cruz Biotechnology Cat# sc-8396, 1:1000) and HuR (Santa Cruz Biotechnology Cat# sc-5261, 1:1000), TTP (Sigma-Aldrich Cat# T5327, 1:1000), Actin- HRP antibody (Santa Cruz Biotechnology Cat# 517582, 1:5000), and tubulin (Santa Cruz Biotechnology Cat# sc-58666, 1:2000) were used for Western blot analysis.
Enhanced chemiluminescence western blot substrate
Manufactured by Thermo Fisher Scientific
Enhanced chemiluminescence Western blot substrate is a reagent used in Western blot analysis. It is designed to produce a luminescent signal proportional to the amount of target protein present in a sample. The substrate reacts with the enzyme-labeled antibodies bound to the target protein, generating a light emission that can be detected and quantified.
Automatically generated - may contain errors
Lab products found in correlation
Actin hrp antibody, by Santa Cruz Biotechnology (1 mentions)
Phospho mek1 2 ser221, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Mabe50, by Merck Group (1 mentions)
Hyperfilm, by Cytiva (1 mentions)
Semi dry blotting system, by Bio-Rad (1 mentions)
4 protocols using enhanced chemiluminescence western blot substrate
1
Western Blot Analysis of FIRRE Overexpression
2
Profiling ERK1/2 and MEK1/2 Phosphorylation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Phosphorylation of ERK1/2 and MEK1/2 were further validated by Western blot analysis. Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (Cat. No. 4320, 1: 1000) and Phospho-MEK1/2 (Ser221) (Car: 2338, 1: 1000) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). The glyceraldehyde-3-phosphate dehydrogenase (ab8245, 1: 5000) antibody was from Abcam, and the HRP-conjugated secondary antibody (Cat. No. SA00001-2, 1: 5000) was from Proteintech. Protein lysates were prepared using RIPA lysis buffer and the concentration was determined using the BCA kit. Then, 25 μg total protein was collected and separated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (EMD Millipore Corp., Billerica, MA, USA). Next, the membranes were blocked using skimmed milk at room temperature for 30 min, washed, and incubated with the primary antibodies at 4°C overnight, and then with the secondary antibody at room temperature for 1 h. The blots were developed using the enhanced chemiluminescence Western blot substrate (Thermo Fisher). Relative protein expression was determined using Image J software.
3
Linc-Pint Regulation in Virus-Infected Cells
4
SDS-PAGE Immunoblotting of PEX5 and MAPK
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For an SDS-PAGE gel either 20 µg of total protein after electroporation or 1.1% of bound and 2.8% of unbound fractions after pull-down binding assays were separated and transferred onto a nitrocellulose membrane (Roth) using a semidry blotting system (Biorad). α-PEX5 (GDA2, rabbit polyclonal) was diluted 1:5,000 in TBS-TX buffer (100 mM Tris, 100 mM NaCl, pH 7.4, 0.1% Triton X®100). To generate these PEX5 antibody pVS1 was expressed in E. coli BL21 (DE3) and recombinant protein was purified under native conditions as described20 (link). The α-MAPK (α-mitogen activated protein kinase) antibody (QIAexpress® Tag 100, Qiagen, against the epitope of MAP kinase 2 (EETARFQPGYRS); mouse monoclonal) was diluted 1:2,000 in TBS-Tw buffer (100 mM Tris, 100 mM NaCl, pH 7.4, 0.02% Tween®20). After using the enhanced chemiluminescence Western Blot substrate (Thermo Scientific) the blot was exposed to film (Hyperfilm; Amersham) for different times and developed.
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!