Anti-Actin (catalog number [Cat#] sc-47778, 1:2,000), anti-p130 (Cat# sc-374521, 1:3,000), anti-p21 (Cat# sc-53870, 1:3,000), anti-p130 (Cat# sc-374521, 1:3,000), and anti-PML (Cat# sc-377390, 1:3,000) were purchased from Santa Cruz Biotechnology. Anti-TAp73 (Cat# A300-126A, 1:1,000) was purchased from Bethyl Laboratories, Inc. Anti-HA (Cat# 901513, 1:2,000) was purchased from BioLegend. Anti-Cleaved Notch1 (Cat# 4741, 1:1,000), anti-Notch1 (Cat# 4380T, 1:1,000), and anti-Hes1 (Cat# 11988S, 1:800) were purchased from Cell Signaling Technology. WesternBright ECL HRP substrate (Cat# K-12043-D20) was purchased from Advansta. Scrambled siRNA (5′-GGC CGA UUG UCA AAU AAU U-3′), sip73α1 siRNA (5′-ACC UGG GGC CCG UGG UUU-3′), siE11 siRNA#1 (5′-GCA CAG UUC GGC AGC UAC A-3′), siE11 siRNA#2 (5′-UCC UCU CGC CCA UGA ACA A-3′), and siNotch1 siRNA (5′-ACA AAG AUA UGC AGA ACA A-3′) were purchased from Horizon Discovery Biosciences Limited. RNAiMax (Cat# 13778150, Invitrogen), Protease Inhibitor Mixture (Cat# 78438), Magnetic Protein A/G beads (Cat# 78609), RevertAid RT Reverse Transcription Kit (Cat# K1691), and DreamTaq DNA Polymerase (Cat# EP0702) were all purchased from ThermoFisher. All reagents were used according to the manufacturer’s protocol.
Magnetic protein a g beads
Manufactured by Thermo Fisher Scientific
Sourced in United States
Magnetic protein A/G beads are a type of bead-based affinity chromatography material used for the purification of antibodies from biological samples. The beads are composed of a magnetic core and a surface coated with either protein A or protein G, which are bacterial proteins that bind to the Fc region of immunoglobulins. These beads can be easily separated from the sample using a magnetic field, allowing for efficient isolation and recovery of the target antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by GE Healthcare (3 mentions)
Igg elution buffer, by Thermo Fisher Scientific (2 mentions)
Human ifn beta duoset elisa kit, by R&D Systems (2 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (2 mentions)
Anti ha antibody, by Cell Signaling Technology (2 mentions)
Rabbit igg isotype control, by Thermo Fisher Scientific (2 mentions)
Edta free protease inhibitor, by Roche (2 mentions)
Phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
Phos tag reagent, by Fujifilm (2 mentions)
Sc 374280 antibody, by Santa Cruz Biotechnology (2 mentions)
Ab195352 antibody, by Abcam (2 mentions)
Complete mini edta free protease inhibitor cocktail, by Roche (2 mentions)
Veriblot reagent, by Abcam (2 mentions)
Dreamtaq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Revertaid rt reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Scrambled sirna, by Horizon Discovery (1 mentions)
Rnaimax, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor mixture, by Thermo Fisher Scientific (1 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
Anti hes1, by Cell Signaling Technology (1 mentions)
19 protocols using magnetic protein a g beads
1
Western Blot Analysis of Notch Signaling
2
ChIP-seq Analysis of Rat Schwann Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ChIP assays were performed with minor modifications43 (link). Purified rat SCs were fixed for 15 min at room temperature with 1% formaldehyde-containing medium. Nuclei were isolated and sonicated in sonication buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA and protease inhibitor cocktail). Sonicated chromatin (~300 µg) was used for immunoprecipitation by incubation with appropriate antibodies (4 μg) overnight at 4 °C. Prerinsed magnetic protein A/G beads (50 μL, Thermo Fisher Scientific, 26162) were added to each ChIP reaction and reactions were incubated for 1 h at 4 °C. The beads were then incubated in 200 ml elution buffer at 65 °C for 20 min to elute immunoprecipitated materials. The ChIP-seq libraries were prepared using NEBNext ChIP-seq Library Prep Master Mix Set for Illumina (NEB catalogue number E6240L) and then run on the Illumina sequencer HiSeq 2500. We used antibodies CTCF (rabbit, Cell Signaling, #3418), H3K27Ac (rabbit, Active motif, 39135), or H3K27me3 (rabbit, Cell Signaling, 9733 s) for ChIP. The crosslinked and sonicated chromatins without immunoprecipitation were used as input controls. For ChIP, real-time PCR was performed using quantitative SYBR green PCR mix (Bio-Rad, 1725121). The values of IgG were normalized to 1.
3
Protein-Protein Interaction Identification via Co-IP
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Co-immunoprecipitation was following a recent report (Chen et al., 2020) (link) with minor modifications. FLAG-tagged VIRP1 with an estrogen-inducible promoter was co-expressed transiently with TAP-tagged IMPa-1 or IMPa-4 via agroinfiltration in N. benthamiana. Three days postinfiltration, 4-mM 17-b-estradiol was infiltrated into leaves 6 h before sampling. The cell lysates from leaf samples were incubated with anti-FLAG antibody (catalog #MA1-142; Thermo Fisher Scientific) for 1 h at 4°C. The magnetic protein A/G beads (catalog #88802; Thermo Fisher Scientific) were then added to the lysate for another 1-h incubation at 4°C with mild shaking. The beads were washed twice with 1 × PBST buffer (137-mM NaCl, 2.7-mM KCl, 10-mM Na2HPO4, 1.8-mM KH2PO4, 0.1% Triton X-100) and once with distilled water. The bound proteins were eluted using IgG elution buffer (Thermo Fisher Scientific) and then subject to immunoblots. Co-immunoprecipitation experiments were repeated twice. For each biological replicate, mixed leaf tissues from three or more plants were used for each treatment.
4
Western Blot Analysis of vIL-6 and IFNβ
5
RNA Immunoprecipitation (RIP) Protocol
6
Phospho-protein Immunoprecipitation and Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoblotting was performed using a standard protocol. Phos-tag reagents were purchased from Wako Chemicals, gels containing phos-tag were prepared according to manufacturer’s instructions. For immunoprecipitations, cells were rinsed twice with ice-cold PBS and lysed in ice-cold lysis buffer (0.15M NaCl, 0.05M Tris-HCl, 0.5% Triton X-100, and one tablet each of EDTA-free protease inhibitors (Roche, 11873580001) and phosphatase inhibitor (Thermo Fisher, 88667) per 50 ml). For immunoprecipitations, primary antibodies were added to the lysates and incubated with rotation overnight at 4°C. 10 μl magnetic protein A/G beads (Thermo Fisher, 88802) were added and incubated for an additional 2 hr. Immunoprecipitates were washed three times with lysis buffer. Immunoprecipitated proteins were denatured by the addition of sample buffer and boiling for 5 mins, resolved by 9% SDS-PAGE, and analyzed via Western blot analysis.
7
Protein Interactome Analysis via Coimmunoprecipitation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CRC cells were lysed in lysis buffer for coimmunoprecipitation assays, and immunoprecipitation was performed using lysates incubated with the indicated antibodies overnight at 4°C and pulled down with magnetic protein A/G beads (Thermo Scientific, USA). After extensive washes in accordance to the manufacturer’s instructions, the antibody-bound proteins were eluted in loading buffer, and the pulled-down protein lysates were analyzed by Western blotting.26 The following primary antibodies were used: anti-LACTB (1:100, CST, USA) and anti-PIK3R3 (1:100, CST, USA).
8
Western Blot Analysis of vIL-6 and IFNβ
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole cell lysates were prepared in SDS-sample buffer (60mM Tris.HCl pH6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.01% bromophenol blue) and separated by SDS-PAGE and then transferred to nitrocellulose membranes (GE Healthcare). The membrane was blocked in PBS-T (PBS with 0.1% Tween20) with 5% nonfat milk (Apex) and then incubated with the indicated primary antibody at 4°C overnight. Please refer to the KEY RESOURCES TABLE for antibodies used in this study. The vIL-6 antibody was purified from the supernatant of v6m 12.1.1 hybridomas (ATCC) using magnetic Protein A/G beads (Thermo Fisher). IFNβ protein in the supernatant were quantified by human IFN-beta DuoSet ELISA kit (R&D Systems) according to the manufacturer’s protocol. IFNβ protein levels (pg/ml) were calculated based on the standard curve generated in the assay.
9
RNA Immunoprecipitation (RIP) Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RIP was performed as previously described (A.M. Khalil et al., 2009 (link)). Briefly, 1×107 cells were lysed in RIP Buffer (150 mM KCl, 25 mM Tris pH 7.4, 5 mM EDTA, 0.5 mM DTT, 0.5% NP-40 substitute) with freshly added RNase inhibitor (100 U/mL) and protease inhibitor by dounce homogenization and centrifuged to pellet insoluble cellular debris. Magnetic Protein A/G beads (ThermoFisher) were pre-washed using magnetic separation with RIP Buffer and incubated with either 10 μg of Rabbit IgG isotype control (ThermoFisher) or 10 μg of anti-HA antibody (Cell Signaling) for 1 hr at 4°C. Antibody-conjugated beads were then washed twice with RIP Buffer to remove any unbound antibody and incubated with lysate for 2 h at RT or overnight at 4°C on a rotator. RIP reactions were then washed five times with RIP Buffer and resuspended in 100 μL of PBS. 10 μL (10%) of the RIP reactions were used for WB analysis against 0.1% input lysate (v/v) to assess RIP efficiency and the remaining 90 μL (90%) was resuspended in TRIzol LS Reagent (ThermoFisher) for RNA extraction and isolation.
10
Immunoprecipitation and Immunoblotting Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in CHAPS buffer [5 mmol/L MgCl2, 137 mmol/L KCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% CHAPS, 10 mmol/L HEPES (pH 7.5)] for 30 min on ice and then clarified by centrifugation at 17,000 g for 10 min at 4°C. Lysates were incubated with an antibody overnight at 4°C. Antigen/antibody complex were captured using magnetic protein A/G beads (Thermo Scientific) for 2 h at 4°C. Unbound proteins were washed three times with 1 mL CHAPS buffer without protease inhibitors. Bound proteins on beads were eluted by incubating in LDS sample buffer for 10 min and were subsequently loaded for immunoblotting.
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!