GST-cPERK, MBP-cPERK, 6His-CNβ, 6His-CNα and 6His-CN-B protein purifications were performed as previously described (Bollo et al., 2010 (link); Li & Sousa, 2012 (link)). All recombinant proteins, even at concentrations of 1.0 to 1.2 mg/ml, showed no absorbance from 310 to 340 nm in absorption spectra (220 to 350 nm), supporting the absence of aggregates in the solution. Recombinant proteins His-CNβ and GST-cPERK (or MBP-cPERK) were incubated in the presence of 0.3 mM DSS cross-linker (#21658; Thermo Scientific,) for 30 min at room temperature. Reactions were quenched in 1 M tris (hydroxymethyl) aminomethane hydrochloride (Tris HCl; pH, 7.5) for 15 min at room temperature. The protein complexes were analyzed by SDS-PAGE (4% to 12%) and detected by immunoblotting either with anti-PERK antibody (PERKUT) or with anti-CNβ (#07–068; Millipore).
Anti cnβ
Manufactured by Merck Group
Anti-CNβ is a laboratory reagent used in research applications. It functions as an antibody that specifically binds to the C-terminal region of the beta subunit of the cyclic nucleotide phosphodiesterase enzyme.
Automatically generated - may contain errors
Lab products found in correlation
Dss crosslinker, by Thermo Fisher Scientific (2 mentions)
Perkut, by Merck Group (2 mentions)
Anti p perk, by Cell Signaling Technology (2 mentions)
Anti actin, by Merck Group (2 mentions)
Anti cnα, by Merck Group (2 mentions)
Anti cn b, by Merck Group (2 mentions)
Anti t perk, by Cell Signaling Technology (2 mentions)
Anti t eif2α, by Cell Signaling Technology (2 mentions)
Anti p eif2α, by Cell Signaling Technology (2 mentions)
Anti gfp, by Abcam (2 mentions)
6 protocols using anti cnβ
1
Purification and Cross-linking of PERK and CN Proteins
2
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analysis was performed using 30 µg of total protein in cell lysates. The following antibodies were used in this study: anti-CNα (#07–067; Millipore), anti-CNβ (#07–068;Millipore), anti-actin (#MAB1501; Millipore), anti-CN-B (#07–069; Millipore), and anti-GFP (#111258; Abcam), anti-P-PERK (#3179; Cell signaling Technology), anti-T-PERK (#3192; Cell signaling Technology), anti-P-eIF2α (#3597; Cell signaling Technology), and anti-T-eIF2α (#9722; Cell signaling Technology).
3
Purification and Cross-linking of PERK and CN Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GST-cPERK, MBP-cPERK, 6His-CNβ, 6His-CNα and 6His-CN-B protein purifications were performed as previously described (Bollo et al., 2010 (link); Li & Sousa, 2012 (link)). All recombinant proteins, even at concentrations of 1.0 to 1.2 mg/ml, showed no absorbance from 310 to 340 nm in absorption spectra (220 to 350 nm), supporting the absence of aggregates in the solution. Recombinant proteins His-CNβ and GST-cPERK (or MBP-cPERK) were incubated in the presence of 0.3 mM DSS cross-linker (#21658; Thermo Scientific,) for 30 min at room temperature. Reactions were quenched in 1 M tris (hydroxymethyl) aminomethane hydrochloride (Tris HCl; pH, 7.5) for 15 min at room temperature. The protein complexes were analyzed by SDS-PAGE (4% to 12%) and detected by immunoblotting either with anti-PERK antibody (PERKUT) or with anti-CNβ (#07–068; Millipore).
4
Immunoprecipitation and Immunoblotting Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were incubated with 0.3 mM DSS for 30 min, and microsomal fractions were obtained as described previously (Bollo et al., 2010 (link)). Anti-CNβ (5 µg) (#07–068; Millipore, (25) was added. After incubation overnight, the immune complex was washed and the eluted proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and detected by immunoblotting with anti-PERK (PERKUT).
5
Immunoprecipitation and Immunoblotting Protocol
6
Western Blot Analysis of Signaling Proteins
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!