Cells were lysed in 100 μl RIPA buffer (MedChemExpress). Proteins were transferred from SDS‒PAGE gels to Immobilon-FLPVDF membranes, blocked, and then incubated with primary antibodies overnight at a 1:1000 dilution. Antibodies against CCR5, AKT, p-AKT, Sp1, CD44, EpCAM, c-Myc, LATS 1, YAP, and GTGF (Abcam) were used. Membranes were then incubated in secondary antibodies, washed, and exposed on a chemiluminescence imaging system (Beijing Sage Creation Science) with ECL (Thermo Fisher Scientific). Western blotting was repeated three times for each band (see Additional file 1 ).
Ripa buffer
Manufactured by MedChemExpress
Sourced in United States
RIPA buffer is a commonly used lysis buffer for the extraction and solubilization of proteins from cells and tissues. It is a detergent-based buffer that helps to disrupt cell membranes and organelles, allowing the release of intracellular proteins. The buffer contains a combination of detergents, salts, and other components that help to maintain the solubility and stability of the extracted proteins.
Automatically generated - may contain errors
Lab products found in correlation
C fos antibody, by Cell Signaling Technology (2 mentions)
Spin column system, by Omega Bio-Tek (2 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
P akt, by Abcam (1 mentions)
C myc, by Abcam (1 mentions)
Epcam, by Abcam (1 mentions)
Anti cleaved parp, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Anti akt, by Merck Group (1 mentions)
Anti bcl 2, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Anti bax, by BioLegend (1 mentions)
Anti pi3k, by Santa Cruz Biotechnology (1 mentions)
Anti p pi3k, by Cell Signaling Technology (1 mentions)
Anti p akt, by Santa Cruz Biotechnology (1 mentions)
Pre stained protein ladder, by Thermo Fisher Scientific (1 mentions)
Bca protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Protease inhibitor, by MedChemExpress (1 mentions)
6 protocols using ripa buffer
1
Comprehensive Protein Expression Analysis
2
Western Blot Analysis of Apoptosis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DA-treated cells were collected and lysed with ice-cold RIPA buffer (MedChemExpress), and a phosphatase inhibitor (Sigma-Aldrich) was added to prevent the degradation of phosphorylated proteins. Forty micrograms of total protein was subsequently separated by SDS‒PAGE and hybridized with the following antibodies after being transferred to PVDF membranes (Merck Millipore) and blocked with blocking buffer (Visual Protein). The primary antibodies were diluted 1:100 and included anti-GAPDH (Abcam, cat. no. ab9484), anti-Bax (Biolegend, cat. no. 633602), anti-Bcl-2 (Cell Signaling, cat. no. 2870s), anti-cleaved-PARP (Cell Signaling, cat. no. 9541s), anti-pPI3K (Cell Signaling, cat. no. 13857s), anti-PI3K (Santa Cruz, cat. no. sc-136208), anti-AKT (Sigma-Aldrich, cat. no. 05-1003), and anti-pAKT (Santa Cruz, cat. no. sc-7985); then, a 1:2,000 dilution of horseradish peroxidase-conjugated secondary antibodies (cat. no. 111-035-003, Jackson ImmunoResearch Laboratories, Inc.) was added and incubated with enhanced chemiluminescence (GE Healthcare Life Sciences) in a Hansor Luminescence Image system (Hansor).
3
Western Blot Protein Quantification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HASMCs and tissue sample proteins were extracted using RIPA buffer containing protease inhibitors (MedChemExpress, NJ, USA). The protein concentration was measured using BCA Protein Assay Reagent (Thermo Fisher Scientific). Lysates were denatured by boiling with SDS-PAGE Buffer loading buffer (Reducing, 5×) (CWBIO, Beijing, China). The proteins and prestained protein ladder (Thermo Fisher Scientific) were resolved on 10% SDS-PAGE gels and transferred to methanol-activated polyvinylidene fluoride membranes (Millipore, MA, USA). Membranes were blocked with 5% nonfat dry milk for regular antibodies, and incubated with primary antibodies overnight at 4 °C. The next day. the membranes were incubated with HRP-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. Detection was performed with Immobilon ECL substrate (Millipore), and the blots were imaged with a ChemiDoc Touch Imaging System (Bio-Rad). Protein expression was quantified using Image Lab 3.0 (Bio-Rad); target protein expression was normalized to that of GAPDH or β-actin in each sample and is expressed as a percentage of the control. The primary antibodies used in the experiments are shown in Table S2 .
4
Chromatin Immunoprecipitation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RBLs were collected as previously described [30 (link)]. Proteins were crosslinked to DNA by adding 270 µL of 37% formaldehyde to 10 mL culture medium (final concentration, 1%). After 10-min incubations at 37 °C, reactions were quenched by adding 0.125 M glycine for 5 min at room temperature. After washing with ice-cold PBS twice, fixed cells were lysed with 1 mL RIPA buffer (MedChem Express, Monmouth Junction, NJ) on ice for 10 min. After centrifugation, nuclei were sonicated (30% amplitude, 20 sets of 15-s bursts) for four rounds. Debris was removed by centrifugation at 13,000 rpm at 4 °C for 10 min; supernatants were transferred to new tubes. Lysates were precleared with protein A/G beads before being incubated with c-Fos antibodies (Cell Signaling Technology, Danvers, MA) or IgG (Santa Cruz, CA) at 4 °C overnight with rotation. The immunocomplex was washed (once each) in low salt, high salt, and LiCl solutions and washed twice in TE buffer. Bead-bound immunocomplexes were eluted with 200 µL elution buffer. To reverse histone-DNA crosslinks, samples were combined with 5 M NaCl and heated at 65 °C for 4 h. DNA product was purified in a spin column system (Omega Bio-Tek, Norcross, GA).
5
Western Blot Analysis of Hippocampus and HT22 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hippocampus or HT22 cells were homogenized in radioimmunoprecipitation assay (RIPA) buffer (MedChemExpress, USA) containing complete protease inhibitor cocktail and phosphatase inhibitor cocktail I and II (MedChemExpress, USA). Western blotting was performed as previously described (Peng et al. 2021 (link)). The involved primary antibodies were listed in Additional file 1 : Table S1.
6
ChIP-seq Protocol for c-Fos Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RBLs were collected as previously described [30] . Proteins were crosslinked to DNA by adding 270 µl of 37% formaldehyde to 10 ml culture medium ( nal concentration, 1%). After 10-min incubations at 37°C, reactions were quenched by adding 0.125 M glycine for 5 min at room temperature. After washing with ice-cold PBS twice, xed cells were lysed with 1 ml RIPA buffer (MedChem Express, Monmouth Junction, NJ) on ice for 10 min. After centrifugation, nuclei were sonicated (30% amplitude, 20 sets of 15-s bursts) for four rounds. Debris was removed by centrifugation at 13,000 rpm at 4°C for 10 min; supernatants were transferred to new tubes. Lysates were precleared with protein A/G beads before being incubated with c-Fos antibodies (Cell Signaling Technology, Danvers, MA) or IgG (Santa Cruz, CA) at 4°C overnight with rotation. The immunocomplex was washed (once each) in low salt, high salt, and LiCl solutions and washed twice in TE buffer. Bead-bound immunocomplexes were eluted with 200 µl elution buffer. To reverse histone-DNA crosslinks, samples were combined with 5 M NaCl and heated at 65°C for 4 h. DNA product was puri ed in a spin column system (Omega Bio-Tek, Norcross, GA).
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!