To study CTHRC1 proteolysis, human plasma was spiked with rhCTHRC1 (5 ng/μL rhCTHRC1, 12% plasma or synovial fluid (SF), 1 x PBS) and incubated at 37°C. Samples were denatured at 97°C in a Laemmli sample buffer and separated on 12% SDS-PAGE. Polyvinylidene fluoride membranes (Merck Millipore) with transferred protein were blocked in 5% (w/v) dry milk for 1 h in PBST and then probed with rabbit antibodies to CTHRC1 (Vli55, www.mmcri.org/antibody ) and were developed with secondary HRP–conjugated goat anti-rabbit (Sigma) antibodies. Enhanced chemiluminescent substrate (Thermo Fisher Scientific) and the BioSpectrum 800 Imaging System (UVP) were used to detect signal.
Biospectrum 800 imaging system
Manufactured by Analytik Jena
Sourced in United States
The BioSpectrum 800 Imaging System is a multi-function imaging platform designed for a variety of scientific applications. It provides high-resolution imaging capabilities for applications such as western blotting, chemiluminescence, fluorescence, and colorimetric detection.
Automatically generated - may contain errors
Lab products found in correlation
Semi dry blotting system, by Bio-Rad (2 mentions)
Enhanced chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Anti akt ab, by Santa Cruz Biotechnology (1 mentions)
Anti erk ab, by Cell Signaling Technology (1 mentions)
Anti p38 ab, by Cell Signaling Technology (1 mentions)
Anti lc3a b, by Abcam (1 mentions)
Anti gapdh ab, by GeneTex (1 mentions)
Anti jnk ab, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Phospho p53 ser20, by Cell Signaling Technology (1 mentions)
Protein assay reagent, by Bio-Rad (1 mentions)
Phospho p38 mapk, by Cell Signaling Technology (1 mentions)
5 protocols using biospectrum 800 imaging system
1
CTHRC1 Proteolysis in Plasma and Synovial Fluid
2
Western Blot Analysis of Platelet Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Platelet proteins were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE gels) and electrophoretically transferred to PVDF membrane by using a semidry blotting system (BioRad). Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline (10 mM Tris-HCl and 150 mM NaCl, pH 8.0) containing 0.05% Tween-20 (TBST) for 1 h at room temperature. Blots were incubated overnight with respective primary antibodies (p-MYPT1, 1:1000; p-MLC, 1:500; anti-RhoA, 1:500; anti-HIF-2α, 1:500; anti-PAI-1, 1:100; anti-pAMPK, 1:1000; anti pACC, 1:1000; anti-β-actin, 1:5000), followed by 3 washings with TBST for 5 min each. Membranes were then placed in HRP-labeled anti-rabbit IgG diluted in blocking buffer or TBST for 1 h. Blots were similarly washed, and antibody binding was detected using enhanced chemiluminescence. Images were acquired on a multispectral imaging system (BioSpectrum 800 Imaging system, UVP) and quantified using VisionWorks LS software (UVP)13 (link).
3
Aloperine Modulation of Autophagy Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were cultured in 10-cm cell culture dishes and treated with aloperine. DMSO was used as a negative control. The whole cellular extract was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the separated proteins were electrically transferred to a PVDF membrane (Millipore Corporation, USA). The membrane was blocked with primary antibodies (anti-LC3 Ab; Abcam, USA; anti-GAPDH Ab; GeneTex, USA; anti-AMPK α-1 Ab; Cell signaling, USA; anti-phosphorylated-AMPK α-1 (Thr 172) Ab; Cell Signaling, USA; anti-Akt Ab; Santa Cruz, USA; anti-phosphorylated-Akt (Ser 473) Ab; Santa Cruz, USA; anti-mTOR Ab; Cell Signaling, USA; anti-phosphorylated-mTOR (Ser 2448) Ab; Cell Signaling, USA; anti-p70S6K Ab; Cell Signaling, USA; anti-phosphorylated-p70S6K (Thr 389) Ab; Cell Signaling; anti-p62/SQSTM1 Ab; Abgent, USA; anti-Erk Ab; Cell Signaling, USA; anti-phosphorylated-Erk (Thr 202/204) Ab; Cell Signaling, USA; anti-JNK Ab; Cell Signaling, USA; anti-phosphorylated-JNK (Thr 183/Tyr 185) Ab; Cell Signaling, USA; anti-p38 Ab; Cell Signaling, USA; and anti-phosphorylated (Thr 180/Tyr 182) Ab; Cell Signaling Ab, USA) and was analyzed using the BioSpectrum 800 Imaging System (UVP, CA, USA).
4
Cyproheptadine's Effects on Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HepG2 and Huh-7 were seeded in 6-well plates at 2 × 105 cells per well and cultured for 24 h, starved in medium without FBS for 24 h, and then treated with 40 μM cyproheptadine for various durations. Total cellular proteins were extracted, and protein concentration was determined for the extracts using the Bio-Rad Protein Assay reagent (Bio-Rad) with bovine serum albumin as a standard. Each lysate (10 μg) was resolved on denaturing polyacrylamide gels and transferred electrophoretically to PVDF transfer membranes. After blocking with 3% blocker (Bio-Rad) in Tris-buffered saline with Tween 20 (TBST), the membranes were incubated at room temperature for 2 h with primary antibodies—1:5000 diluted antibody against GAPDH; 1:1000 diluted antibody against PARP, p21, p27, Rb (D20), phospho-Rb (Ser795), cyclin D1, p38 MAPK, phospho-p38 MAPK (Thr180/Tyr182), CHK2, phospho-CHK2 (Thr68), p53 (7F5), or phospho-p53 (Ser20) (Cell Signaling, Danvers, MA); or 1:1000 diluted antibody against p16INK4A or HBP1 (Millipore, Temecula, CA). Immunoreactive proteins were detected by incubation with horseradish peroxidase–conjugated secondary antibodies for 1 h at room temperature. After washing with TBST, the reactive bands were developed with an enhanced chemiluminescent HRP substrate detection kit (Millipore, Billerica, MA) and identified using the BioSpectrum 800 imaging system (UVP).
5
SDS-PAGE Protein Separation and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Platelet proteins were separated on 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gels and electrophoretically transferred to the PVDF membrane by using a semidry blotting system (Bio-Rad). Membranes were blocked with 5% nonfat dry milk or BSA in Tris-buffered saline (10 mM Tris-HCl and 150 mMNaCl; pH 8.0) containing 0.05% Tween-20 (TBST) for 1 hour at room temperature. Blots were incubated overnight with respective primary antibodies (anti-Shh 1:1000; anti-pAMPK, 1:1000 in 5% BSA; anti-AMPK, 1:1000 in 5% BSA; anti-pACC, 1:1000 in 5% BSA; anti-pMLC2, 1:500 in 5% BSA; anti-MLC2, 1:1000; anti-pMYPT, 1:1000 in 5% BSA; anti–β-actin, 1:5000), followed by 3 washings with TBST for 5 minutes each. Membranes were then placed in HRP-labeled anti-rabbit or anti-mouse IgG diluted in blocking buffer or TBST for 1 hour. Blots were similarly washed, and antibody binding was detected using enhanced chemiluminescence. Images were acquired on a multispectral imaging system (BioSpectrum 800 Imaging system, UVP) and quantified using VisionWorks LS software (UVP).24 (link)
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!