The BV-2 cells were collected and treated with radioimmunoprecipitation (RIPA, Boster Biological Technology Co., Ltd, Wuhan, China) lysis buffer and measured using a bicinchoninic acid (BCA, Boster Biological Technology Co., Ltd, Wuhan, China) kit as previously described [34 ]. Equal amounts of protein were loaded on an SDS polyacrylamide gel, electrophoresed, then transferred to polyvinylidene fluoride (PVDF, Millipore, Saiwen Biotechnology Co., Ltd, Beijing, China) membranes, which were then overnight incubated with primary antibodies (anti-Nrf2 (1:1000), anti-Trem1 (1:1000), anti-Trem2 (1:1000), anti-CD80 (1:1000), anti-CD206 (1:1000), anti-TNF-α (1:2000, ab6671, aBCAm), and anti-BDNF (1:1000, ab108319, aBCAm), β-actin (1:10000, AP0060, Biogot technology Co., Ltd, Nanjing, China), additional antibody information as previously described) at 4 °C. The membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (Goat Anti Rabbit IgG (1: 1000, ab6702, aBCAm), Goat Anti-Rat IgG (1:5000, ab97057, aBCAm), Goat Anti-Armenian Hamster IgG (1:5000, ab5738, aBCAm)) and visualized through chemiluminescence (ECL chemiluminescence reagent kit, Boster Biological Technology Co., Ltd, Wuhan, China). Densitometry was performed to quantify the signal intensity using Image J software (https://imagej.net ).
Ab6702
Manufactured by Abcam
Sourced in United Kingdom, China, United States
Ab6702 is a primary antibody that binds to a specific target protein. It is intended for use in various laboratory applications, such as immunoassays and immunohistochemistry. The core function of this product is to provide a specific and reliable detection of the target protein.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (6 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (5 mentions)
Phenol chloroform isoamilic acid, by Merck Group (4 mentions)
Bioruptor pico, by Diagenode (4 mentions)
Ab32381, by Abcam (4 mentions)
Ez magna rip kit, by Merck Group (4 mentions)
Ab290, by Abcam (3 mentions)
Tri reagent, by Merck Group (3 mentions)
Pab 195 050, by Abcam (2 mentions)
Pab0923 p, by Diagenode (2 mentions)
Mab 081 100, by Abcam (2 mentions)
Protein g dynabead, by Thermo Fisher Scientific (2 mentions)
Dnase, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ab6671, by Abcam (1 mentions)
Ab97057, by Abcam (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Radioimmunoprecipitation assay (ripa), by Boster Bio (1 mentions)
Bicinchoninic acid (bca), by Boster Bio (1 mentions)
Ab108319, by Abcam (1 mentions)
20 protocols using ab6702
1
Western Blot Analysis of Microglial Markers
2
ChIP Assay for Sp1 Binding on UCHL1 Promoter
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ChIP assay was performed using the ChIP kit (Abcam). Briefly, 1% formaldehyde was added to HEI-OC1 cells for 10 min at room temperature. The fixed cells were washed twice with phosphate-buffered saline and were lysed using a lysis buffer (0.1% SDS, 0.5% Triton X-100, 20 mM Tris-HCl, pH 8.1) that contained 150 mM NaCl and a protease inhibitor, after which chromatin fragments were obtained using sonication using a 10 sec on and 10 sec off mode for 12 cycles at 4˚C. Following centrifugation at 13,000 x g for 10 min at 4˚C, the DNA fragments were incubated with antibodies against Sp1 (cat. no. ab227383; 1:200: Abcam) or IgG (cat. no. ab6702; 1:40; Abcam) for 2 h at 4˚C. The abundance of Sp1 on the UCHL1 promoter was measured by PCR as aforementioned. The sequence of oligonucleotides flagging the Sp1 binding site in the UCHL1 promoter was 5'-CCCGCCCCC-3'.
3
ChIP-qPCR: Mapping Transcription Factor Binding
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Chromatin immunoprecipitation (ChIP) assays were performed using anti-GFP (Abcam ab290), anti-LHP1 (Covalab pab0923-P), anti-H3K27me3 (Diagenode pab-195-050), and anti-IgG (Abcam ab6702), as described in Ariel et al. [52 (link)]. Crosslinked chromatin was sonicated using a water bath Bioruptor Pico (Diagenode; 30 s ON/30 s OFF pulses; 10 cycles; high intensity). ChIP was performed using Invitrogen Protein A Dynabeads. Precipitated DNA was recovered using Phenol:Chloroform:Isoamilic Acid (25:24:1; Sigma) and subjected to RT-qPCR (primers used are listed in Additional file 2 : Table S4). Untreated sonicated chromatin was processed in parallel and considered as the input sample.
4
Quantitative Protein Analysis of Retinal Tissues
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RB tissues and normal retinal tissues or cells were lysed to isolate total protein. Proteins were separated by 10% sodium dodecyl sulfide-polyacrylamide gel electrophoresis (SDS-PAGE), and then electrotransferred to nitrocellulose membrane (Millipore, Temecula, CA, USA) and blocked with 5% BSA at room temperature for 2 h to block nonspecific binding. Primary rabbit anti-p38 MAPK, anti-P-p38 MAPK, USP22, SIRT1, SOST, and GAPDH (ab128915; 1: 10000, Abcam) were incubated with the membrane overnight at 4°C. The membrane was then incubated with HRP-labeled goat anti-rabbit secondary antibody (ab6702; 1:2000, Abcam) for 1 h at room temperature, developed using ECL working solution (EMD Millipore, USA) and exposed. Image J analysis software was used to quantify the gray scale of each band in the Western blot Image. GAPDH was taken as internal reference.
5
Argonaute-2 RIP Assay Protocol
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An RNA-binding protein immunoprecipitation (RIP) assay was performed using the EZ-magna RIP kit (Millipore, China) according to the manufacturer's instructions. First, A2780 and SKOV3 cells were harvested and lysed in complete RIP lysis buffer. Then, the cell extracts were incubated with RIP buffer containing anti-Argonaute-2 antibody-conjugated magnetic beads (ab32381, Abcam, Shanghai, China); an anti-immunoglobulin G (anti-IgG) antibody (ab6702, Abcam) was used as a control. Then, the samples were incubated with protease K and oscillated to digest proteins and isolate immunoprecipitated RNAs. The RNA concentration was determined by using a NanoDrop spectrophotometer, and the purified RNAs were evaluated by RT-PCR.
6
Methylated DNA Immunoprecipitation for Epigenomics
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Methylated DNA immunoprecipitation (MedIP) assays were performed using anti 5-mC (Diagenode Mab-081-100) and anti-IgG (Abcam ab6702), as described in Nagymihály et al. [130 (link)]. Genomic DNA (1 μg) was sonicated using a water bath Bioruptor Pico (Diagenode; 30 s on/30 s off pulses; 4 cycles; high intensity). MedIP was performed using Invitrogen Protein G Dynabeads. Precipitated DNA was recovered using Phenol:Chloroform:Isoamilic Acid (25:24:1; Sigma) and subjected to RT-qPCR (primers used are listed in Additional file 2 : Table S4). Untreated sonicated genomic DNA was processed in parallel and considered as the input sample.
7
Western Blot Analysis of Protein Expression
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RIPA lysis buffer (Wanlei Bio, China) containing protease inhibitor cocktail (Roche) was used to extract the protein from the harvested cells. Then, the protein samples were loaded onto 10% SDS-PAGE gels and transferred onto PVDF membranes (Millipore, USA). After blocking with 5% BSA, the PVDF membranes were incubated with primary antibodies overnight at 4°C and probed with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature. Thereafter, enhanced chemiluminescence (ECL) (Wanlei Bio, China) was used to detect the protein bands. The following antibodies were included: GAPDH (WL01114, 1:2000 dilution, Wanlei Bio, China); TRIM27 (ab154931, 1:500 dilution, Abcam, USA); Akt (ab179463, 1:1000 dilution, Abcam, USA); p-Akt (ab192623, 1:1000 dilution, Abcam, USA); Goat Anti-mouse IgG H&L (HRP) (ab205719, 1:2000 dilution, Abcam, USA) and Goat Anti-Rabbit IgG H&L (HRP) secondary antibody (ab6702, 1:2000 dilution, Abcam, USA).
8
RIP Assay for AGO2 Binding
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Following the product specifications, we adopted the EZ-magna RIP kit (Millipore, United States) to perform the RIP assay. HB611 and HuH-7 cells were collected and lysed in a full RIP lysis buffer. Cell extracts were incubated with RIP buffer containing magnetic beads conjugated to human AGO2 antibodies (ab32381, Abcam, Cambridge, United Kingdom); we used the IgG antibody (ab6702, Abcam) as control. Samples were incubated with protease K, and oscillated to digest the protein and isolate the immunoprecipitated RNA. Using a NanoDrop spectrophotometer, we measured the concentration of RNA and performed real-time PCR analysis using the purified RNA.
9
RNA Immunoprecipitation in Plants
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RNA immunoprecipitation (RIP) assays were performed on transiently transformed N. benthamiana leaves as described in Sorenson and Bailey-Serres [129 (link)], or in 2-week-old A. thaliana OE VIM1.1 seedlings as described in Bardou et al. [70 (link)], using anti-GFP (Abcam ab290) and anti-IgG (Abcam ab6702). RIP was performed using Invitrogen Protein A Dynabeads. Precipitated RNAs were prepared using TRI Reagent (Sigma-Aldrich), treated with DNaseI (Fermentas) and subjected to RT-qPCR (High Capacity cDNA Reverse Transcription Kit (Thermo); primers used are listed in Additional file 2 : Table S4). Total RNAs were processed in parallel and considered as the input sample.
10
RNA Immunoprecipitation Assay for AGO2
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The EZ-magna RIP kit (Millipore, United States) was applied to carry out the RIP assay according to the product specifications. First, the HepG2 cells were collected and lysed in a full RIP lysis buffer. Cell extracts were then incubated with RIP buffer containing magnetic beads conjugated to human AGO2 antibodies (ab32381, abcam, Cambridge, United Kingdom), and IgG antibody (ab6702, abcam, Cambridge, United Kingdom) was used as controls. The samples were incubated with protease K and oscillated to digest the protein and isolate the immunoprecipitated RNA. The concentration of RNA was then measured using a NanoDrop spectrophotometer and real-time PCR analysis of the purified RNA was performed.
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