For protein analysis, total protein was obtained from a pool of whole brain homogenates (n=9) per genotype. Two biological replicates per genotype were collected. Brains were excised and stored for protein isolation in 1X Cell Lysis Buffer (ThermoFisher Scientific) with protease inhibitors cocktail (ThermoFisher Scientific). DNA was isolated from the tail tissue for genotyping and protein was isolated from brain homogenates using manual homogenization. Protein quantification was performed using Precision Red Protein Assay Reagent (Cytoskeleton) according to manufacturer’s instructions. Samples were then sent to the Biomolecule Analysis and Omics Unit (BAOU) at The University of Texas El Paso for sample processing and proteomic analysis.
Precision red protein assay reagent
Manufactured by Cytoskeleton
Sourced in United States
The Precision Red Protein Assay Reagent is a colorimetric assay used for the quantitative determination of total protein concentration in a sample. The reagent contains a red dye that binds to proteins, resulting in a color change that can be measured using a spectrophotometer. The intensity of the color is proportional to the protein concentration in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitors cocktail, by Thermo Fisher Scientific (1 mentions)
Mab1501, by Merck Group (1 mentions)
G1544, by Merck Group (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Luminata forte western hrp substrate, by Merck Group (1 mentions)
Las 4000 plus, by Fujifilm (1 mentions)
Las 1000plus, by Fujifilm (1 mentions)
G lisa activation assay, by Cytoskeleton (1 mentions)
Signal seeker acetyl lysine detection kit, by Cytoskeleton (1 mentions)
Bead elution buffer, by Cytoskeleton (1 mentions)
Blastr lysis buffer, by Cytoskeleton (1 mentions)
Blastr filter system, by Cytoskeleton (1 mentions)
6 protocols using precision red protein assay reagent
1
Protein Isolation and Quantification from Whole Brain
2
Analyzing Protein Expression in C. elegans
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein lysates were prepared from age-matched animals grown on E. coli HT115(DE3) carrying control RNAi vector or cacn-1 RNAi vector as follows: Animals were washed from plates using M9 with 0.1% Tween and allowed to settle. After washing, worm cuticles were disrupted by chopping with a razor blade. Animals were resuspended in cold lysis buffer (10 mM Tris pH 8.8, 150 mM NaCl, 0.5 mM EDTA, 0.05% NP40) containing protease inhibitors (Halt Protease Inhibitor Cocktail, Thermo Scientific; Rockford, IL, USA). Protein extracts were obtained by Dounce homogenization for 50–100 strokes on ice. Lysates were clarified by centrifuging for 5 minutes at 20,000 g. The protein in the supernatant was quantified using the Precision Red Protein Assay Reagent (Cytoskeleton; Denver, CO, USA). Samples were loaded onto Mini-PROTEAN TGX precast 10% polyacrylamide gels (Bio-Rad; Hercules, CA, USA) for SDS-PAGE. Antibodies used for Western blotting are anti-POP-1 at 1∶1000 (94I, gift from R. Lin), anti-GFP at 1∶1000 (G1544, Sigma), and anti-Actin at 1∶2000 (MAB1501, Millipore). Secondary antibodies used were goat anti-rabbit (PI31460, Thermo Scientific Pierce) and donkey anti-mouse (SA1100, Thermo Scientific Pierce).
3
Cellular Senescence Biomarker Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Senescence-associated beta-galactosidase (β-gal) was analyzed using colorimetric β-gal staining kit (Cell Signaling) or quantified by fluorometric kit (Cell Biolabs). Total protein was measured using Precision Red protein assay reagent (Cytoskeleton, Inc.).
4
Protein Expression and Signaling Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following stimulation, fibroblasts were lysed with RIPA buffer, and the proteins were extracted from the cells and quantified using Precision Red Protein Assay Reagent (Cytoskeleton, Denver, CO). Equal amounts of protein were separated in an 8% to 12% SDS-PAGE gel followed by transfer to methanol-preactivated polyvinylidene difluoride membranes (Millipore, Billerica, MA) for 50 minutes. The membranes were then blocked in Immuno-Block buffer (KAC, Hyogo, Japan.) for 1 hour at room temperature and incubated with primary antibodies against MMP-2 (1:1000 dilution), MMP-9 (1:2000 dilution), phosphorylated FGFR1 (1:500 dilution), STAT1 (1:500 dilution), STAT3 (1:1000 dilution), JAK1 (1:500 dilution), JAK2 (1:500 dilution), JAK3 (1:500 dilution), unphosphorylated FGFR1, STAT1, STAT3, JAK1, JAK2 and JAK3 (each 1:1000 dilution) overnight at 4°C, according to the manufacturer’s instructions. After washing, the membranes were developed with Luminata Forte Western HRP substrate (Millipore, Billerica, MA) for detection and visualized using Fujifilm LAS-4000 Plus (Fujifilm). The intensity of bands was quantified using the software program ImageJ Gauge (LAS-1000plus, Fujifilm) to allow correction for protein loading.
5
Rac1, RhoA, and Cdc42 GTPase Activation Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
G-LISA Activation Assays (Cytoskeleton, Inc., Denver, CO, USA) were used to compare the levels of activated Rac1 (BK128), RhoA (BK124), and Cdc42 (BK127) GTPases in different groups of cells after LPA and PMA stimulations. Lysates were prepared according to the protocol, except for lysates for G-LISA Cdc42 Activation Assay, in which lysis buffer was a mixture of GL35:GL36 buffers in ratio 1:1. This was done upon an advice of technical support of Cytoskeleton, Inc. The Precision Red Protein Assay reagent was used for quantification of protein concentration in lysates, which was around 0.5 mg/mL. Active proteins were detected by incubation with primary antibody detecting given GTPase and appropriate secondary antibody conjugated with horseradish peroxidase (HRP). Colorimetric signal was measured at 490 nm using a microplate spectrophotometer. The data were presented as ratio between stimulated and nonstimulated (starved for 24 h) cells with normalization to the control cells.
6
Acetyl-Lysine Detection in Muscle
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Signal-Seeker™ Acetyl-Lysine detection kit (Cytoskeleton, CO, USA) was used to measure SERCA1a acetylation and SERCA2a acetylation in gastrocnemius muscle, according to the manufacturer's protocol and as previously described (Oldfield et al. 2021) (link). Gastrocnemius muscle tissue was removed from -80°C and immediately lysed with ice-cold BlastR™ lysis buffer (Cytoskeleton, CO, USA) and DNA was removed from the lysate using the BlastR™ filter system (Cytoskeleton, CO, USA). Following dilution with BlastR™ dilution buffer (Cytoskeleton, CO, USA), the protein concentration of the lysate was determined with the Precision Red™ Protein Assay Reagent (Cytoskeleton, CO, USA) and assessed at 600 nm. The sample was then immunoprecipitated by incubating the lysate with acetylated-lysine affinity beads (Cytoskeleton, CO, USA) overnight at 4°C with rotation. The beads were pelleted and washed 3 times with BlastR™ wash buffer and bound acetylated-lysine containing proteins were eluted using bead elution buffer (Cytoskeleton, CO, USA
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!