Proteins were extracted using RNA immunoprecipitation (RIP) assay lysis buffer (Beyotime, Beijing, China) and quantified by bicinchoninic acid (BAC) method, then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and shifted onto a polyvinylidene fluoride (PVDF) membrane. Immunoblotting used antibodies against CD9 (1: 5000, ab68418, Abcam, Cambridge, MA, USA), CD63 (1: 2000, ab68418, Abcam), E-cadherin (E-cad; 1: 1000, ab15148, Abcam), vimentin (1: 5000, ab92547, Abcam), ERα (1: 3000, ab13504, Abcam), GAPDH (1: 10 000, ab181602, Abcam) and the secondary horseradish peroxidase (HRP)-conjugated antibody (1: 1000, ab9482, Abcam). The protein bands were visualized using electrochemiluminescence (ECL).
Rip assay lysis buffer
Manufactured by Beyotime
Sourced in China
The RIP assay lysis buffer is a solution used in the preparation of cell lysates for RNA immunoprecipitation (RIP) assays. The buffer is designed to efficiently extract and solubilize cellular proteins and RNA complexes, while maintaining their interactions and native conformation. It provides the necessary conditions for the immunoprecipitation of ribonucleoprotein complexes, which is a critical step in the RIP assay procedure.
Automatically generated - may contain errors
Lab products found in correlation
Ab181602, by Abcam (2 mentions)
Rip kit, by Merck Group (2 mentions)
Ab9482, by Abcam (1 mentions)
Ab15148, by Abcam (1 mentions)
Ab68418, by Abcam (1 mentions)
Ab13504, by Abcam (1 mentions)
Ab92547, by Abcam (1 mentions)
Bradford protein assay kit, by Beyotime (1 mentions)
Polyvinylidene difluoride membrane, by Beyotime (1 mentions)
Ab182408, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Ab2410, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Enhanced ecl detection kit, by Beyotime (1 mentions)
β actin, by Abcam (1 mentions)
Mouse anti tnfaip2 antibody, by Santa Cruz Biotechnology (1 mentions)
Ab450, by Abcam (1 mentions)
Ab172730, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
7 protocols using rip assay lysis buffer
1
Protein Extraction and Immunoblotting Analysis
2
Western Blot Analysis of IGF-1R Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RIP assay lysis buffer (Beyotime Biotechnology Ltd.) was used to extract total protein. Protein quantification was performed using the Bradford Protein Assay Kit (Beyotime Biotechnology Ltd.,). Equivalent amounts of protein were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins were transferred onto polyvinylidene difluoride membranes (Beyotime Biotechnology Ltd.) and blocked with 5% skimmed milk. The membranes were then incubated with primary antibodies against IGF-1R (ab182408; 1:1000 dilution; Abcam, Cambridge, MA, USA) or GAPDH (ab181602; 1:1000 dilution; Abcam) overnight at 4 °C. The membranes were incubated with horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (ab205718; 1:5000 dilution; Abcam) followed by detection with the BeyoECL moon protein detection kit (Beyotime Biotechnology Ltd.). GAPDH was used as a loading control.
3
miR-19b Binds to CCL1 in RIP Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The binding between miR-19b and CCL1 was detected using RIP kit (Millipore, Temecula, CA, USA). Cells were ice-bathed using an equal volume of RIP assay lysis buffer (Beyotime), and then centrifuged for 10 minutes at 14,000 rpm (4°C) to remove the supernatant. A part of the cell extract was used as input, while the rest was utilized for co-precipitation reaction with Ago2 antibody (ab32381, 1: 100; Abcam). IgG antibody (ab2410, 1: 100; Abcam) was utilized as a control. RNA was extracted from the samples with TRIzol reagent (Invitrogen), and then the enrichment was analyzed by RT-qPCR.
4
Western Blot Analysis of TNFRSF21 in LUAD
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RIP assay lysis buffer (Beyotime, Shanghai, China) was used to extract the protein from LUAD cells or tissues. After measuring the density of all proteins, the target proteins (30 µg/lane) were separated by SDS-PAGE (10%) and then carefully transferred onto polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA). Later on, the transferred membranes were blocked with silk milk (5%) for 1 h at 37°C and incubated with the primary anti-TNFRSF21 (catalogue no. ab8417; 1:800; Abcam; USA) and β-actin (catalog no. ab8226; 1:3,000; Abcam; USA) overnight at 4°C. On the second day, the membranes were then treated with a corresponding secondary antibody (catalog no. ab6721; 1:5,000; catalog no. ab6728; 1:5,000; Abcam; USA). Finally, the protein signals on the membrane were visualized by enhanced ECL detection kit (Beyotime, Shanghai, China).
5
Protein Extraction and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extraction was conducted by radioimmunoprecipitation (RIP) assay lysis buffer (Beyotime, China). The protein specimens were electrophoretically separated on a 10% SDS-PAGE gel. The proteins were then transported onto nitrocellulose filter membranes and detected by the corresponding antibodies. The antibodies and the corresponding dilutions used for western blotting were as follows: mouse anti-TNFAIP2 antibody (Santa Cruz, USA; 1:500) and β-actin rabbit mAb (CST, USA; 1:1000).
6
RNA Immunoprecipitation Assay for LINC01093-FOXP3 Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The RIP kit (Millipore, Bedford, MA, USA) was applied to detect the binding of LINC01093 and FOXP3. The cells were washed by pre-cooled PBS, after which the supernatant was discarded. Next, the cells were lysed by RIP assay lysis buffer (P0013B, Beyotime) of equal volume in an ice bath for 5 min, and centrifuged at 12,000 × g for 10 min at 4°C, with supernatant obtained. One part of the cell extract was used as input, and the other was co-precipitated with antibodies FOXP3 (ab450, 2 μg/mL of cells, Abcam) and immunoglobulin G (IgG) (ab172730, 1:100, Abcam), which was used as NC. A total of 50 μL of magnetic beads from each co-precipitation reaction system were washed, resuspended in 100 μL of RIP wash buffer, and incubated with 5 μg antibodies for binding. The magnetic bead-antibody complex was then washed, resuspended in 900 μL of RIP wash buffer, and incubated with 100 μL of cell extract at 4°C overnight. The samples were placed on a magnetic base to collect the magnetic bead-antibody complex, after which the samples and the input were detached by protease K to extract RNA, which was used for qRT-PCR.
7
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from different treated cells using RIP assay lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with protease inhibitor and the protein concentration was measured by BCA Protein Assay Kit (Pierce, Appleton, WI). Then, a total of 30 μg protein per well were separated by 10% SDS-PAGE and transferring onto a PVDF membrane (Millipore, Billerica, MA), and the separated membrane was blocked by the use of 5% nonfat milk solution for 1 h followed by an overnight incubation with primary antibodies against Bcl-2, Bax, cleaved caspase3, LC3B, ATG7, Beclin1 and GAPDH (1:1000, Santa Cruz, USA) at 4°C. At the end of three washes, the membrane was subjected to incubation for another 1 h using horseradish peroxidase-conjugated secondary antibody. Visualization of the bands was carried out using the enhanced chemiluminescence (ECL) system (Beyotime, Shanghai, China) and quantification using Image J (NIH, USA) [29 (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!