To detect the interaction between GSK-3β and Bax, about 4 ml of GSK-3β or Bax antibodies respectively were firstly added to 400 ml cell lysates. According to the manufacturer’s protocol, the mixtures were mixed on a rocker at ambient temperature for 2 h. The immunocomplexes were captured by the addition of protein A-agarose (Roche Applied Sciences, Indianpolis, Cat. No. 11 134 515 001) mixed at 1:10 ratio, followed by incubation at ambient temperature for 1 h. The beads were washed by Washing Buffer 1, Washing Buffer 2, Washing Buffer 3 (Roche Applied Sciences, Indianpolis, Cat. No. 11 134 515 001) and then collected by centrifugation at 12,000 rpm for 30 s. After the final wash, the beads were mixed with 60 ml of 2× Laemmli sample buffer, heated at 98 °C for 8 min, and analyzed by Western blotting using GSK-3β, Bax6A7 or Bax antibody (Cell Signaling Technology, Shanghai, China).
Protein a agarose
Manufactured by Roche
Sourced in United States, Switzerland, Germany
Protein A agarose is a chromatography resin consisting of Protein A, a bacterial protein that binds to the Fc region of immunoglobulins, immobilized on an agarose matrix. It is commonly used for the purification and isolation of antibodies and antibody-containing samples.
Automatically generated - may contain errors
Lab products found in correlation
Qiaquick pcr purification kit, by Qiagen (5 mentions)
Qiaquick spin kit, by Qiagen (4 mentions)
Anti γ catenin, by BD (3 mentions)
Anti β catenin, by BD (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Ab5131, by Abcam (3 mentions)
Complete protease inhibitor cocktail, by Roche (3 mentions)
Protein g agarose, by Merck Group (3 mentions)
Protein g agarose, by Roche (2 mentions)
Pcr purification kit, by Qiagen (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Bioruptor, by Diagenode (2 mentions)
Normal rabbit igg, by Cell Signaling Technology (2 mentions)
Bethyl a302 130a, by Fortis Life Sciences (2 mentions)
Gradient sds page gel, by Thermo Fisher Scientific (2 mentions)
Cbp p300, by Santa Cruz Biotechnology (2 mentions)
Anti α tubulin clone cp06, by Calbiotech (2 mentions)
M2 conjugated agarose beads, by Merck Group (2 mentions)
Baf155 antibody, by Cell Signaling Technology (2 mentions)
Mg132, by Avantor (2 mentions)
78 protocols using protein a agarose
1
GSK-3β and Bax Interaction Assay
2
Antibody Immunoprecipitation Protocol
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Immunoprecipitation was carried out as described earlier 29 . In brief, cells were lysed for 1 hr at 4°C in a lysis buffer. The cell lysates were precleared with 10% (vol/vol) protein A‐agarose (Roche, Branford, CT, USA) for 1 hr on a rocking platform. Specific antibodies were added and rocked for overnight. Immunoprecipitates were captured with 10% (vol/vol) protein A‐agarose for another hour. The agarose beads were spun down and washed thrice with NET buffer. The antigens were released and denatured by adding SDS sample buffer.
3
Immunoprecipitation Protocol for Protein Analysis
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Immunoprecipitation was carried out as we described [50 (link)]. In brief, cells were lysed for 1 h at 4°C in a lysis buffer. To perform immunoprecipitation, the cell lysates were precleared with 10% (vol/vol) protein A-agarose (Roche, Branford, CT, USA) for 1 h on a rocking platform. Specific antibodies were added and rocked for overnight. Immunoprecipitates were captured with 10% (vol/vol) protein A-agarose for another hour. The agarose beads were spun down and washed thrice with NET buffer. The antigens were released and denatured by adding SDS sample buffer.
4
Coimmunoprecipitation Assay for Protein-Protein Interactions
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For immunoprecipitation, the following antibodies were used: KDM4B (Cell Signaling; #8639, 1:200 dilution), eIF2α (Santa Cruz; sc-133132, 1:200 dilution), PERK (Cell Signaling; #5683, 1:200 dilution), and P-eIF2α (Cell Signaling; #3398, 1:200 dilution). Coimmunoprecipitation assays were performed as described previously (Wee et al., 2015 (link)) with a modified protocol using NETN buffer (25 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 10% glycerol, and 0.3% NP-40) containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche Diagnostics) to extract whole-cell protein, or a cell fractionation protocol for cytoplasmic and nuclear fractionation. Cell lysates were incubated with the indicated antibodies at 4°C overnight. The protein complex was captured using protein A–agarose (for rabbit primary antibody) or protein G–agarose (for mouse primary antibody) beads (Roche Diagnostics) at 4°C for 4 h, and agarose beads were collected by centrifuge and washed three times with washing buffer. The precipitated proteins were dissolved in SDS sample buffer along with 3 mM dithiothreitol and subjected to Western blot analysis.
5
Immunoblot and Immunoprecipitation of MYC2 Protein
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Immunoblot and immunoprecipitation assays were performed as previously described with slight modifications (54 (link)). Briefly, 6-day-old seedlings were treated with 100 μM JA for 6 h, and then roots were collected and ground in liquid nitrogen. The CK2mut seedlings were pre-treated with 1 μM Dex for 24 h before JA treatment. Proteins were extracted in RIPA buffer [50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 10 mM NaF, 10 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 mM PMSF, 5 μg/mL leupeptin, 1 μg/mL aprotinin, 1 μg/mL pepstatin]. Anti-GFP antibody (Santa Cruz) was used to detect MYC2-GFP proteins. For immunoprecipitation assays, protein extracts were pre-incubated for 1 h at 4°C with Protein A agarose (Roche), and then incubated for 4 h at 4°C with Anti-GFP agarose conjugate (Santa Cruz). Immunocomplexes were washed five times with RIPA buffer and eluted by adding 4 × SDS loading buffer followed by 5 min of boiling. The anti-phospho-(Ser/Thr) antibody (Abcam) was used to detect the phosphorylated isoform of MYC2. All the experiments were performed three times and showed similar results.
6
Immunoprecipitation and Immunoblotting Analysis
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Immunoprecipitation and imunoblotting were performed as described previously [26 (link)]. The reagents were as following: anti-β-catenin (BD Bioscience, Cat# 610153), anti-γ-catenin (BD Bioscience, Cat# 610253), anti-CBP (Santa Cruz, clone SC-369), anti-p300 (Santa Cruz, clone SC-584), anti-activated- β-catenin (Millipore, clone 8E7), anti-lamin A/C (Santa Cruz, SC-7293), NE-PER Nuclear extraction reagent (Pierce, Cat#78833), Protease inhibitor cocktail (Calbiochem, Cat#539137), Protein A-agarose (Roche, Cat#11134515001), Illustra microspin columns (GE Healthcare, Cat#27-3565-01), ECL Plus (GE Healthcare, Cat#RPN 2132), and Blue ultra autorad film (BioExpress, Cat# F9029-8X10).
7
ChIP-seq analysis of HA-tagged protein
8
Immunoprecipitation and Ubiquitination Assay Protocol
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For immunoprecipitation, Aag2 cells were transfected with indicated plasmids and suspended in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 1% Nonidet P40, 0.5% sodium deoxycholate, 150 mM sodium chloride, 1 tablet complete protein inhibitor cocktail/50 mL, and 0.7 μg/mL pepstatin. To reduce the nonspecific binding of unrelated proteins to protein A-agarose (Roche), a 50-μL suspension of homogeneous agarose beads was mixed with the sample, incubated at least 3 h at 2°C to 8°C, and then centrifuged for 20 s at 12,000 × g. The supernatant was separated and incubated with specific antibodies at 4°C overnight. The beads with complex were pulled down and washed with lysis buffer, washing buffer 2, and washing buffer 3, according to the producer’s instructions. After centrifugation, the agarose beads were resuspended in 2× SDS loading buffer, and proteins were heat denatured. After removal of the agarose, the mixture was subjected to immunoblotting with different antibodies (all antibodies used are listed in Table S6). Ubiquitination assays were performed as described elsewhere (53 (link)). After homogenizing in lysis buffer, the tissue samples were centrifuged. The supernatant was mixed with SDS sample buffer and then heated before immunoblotting with anti-ubiquitin antibodies.
9
Analyzing HDAC5 Binding in Neurons
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Hippocampal primary neurons (DIV4) were transfected with Myc-HDAC5 using lipofectamine 2000 reagent (Invitrogen). Neurons and tissues were fixed with 18.5% (w/v) paraformaldehyde in PBS and sonicated. The sonicated samples were rotated with protein A agarose (Roche Applied Sciences) for 1 h at 4 °C to preclear them. Antibodies were added to the sonicated samples and incubated overnight at 4 °C. The antibodies used are given in Table S2 . Immunoprecipitated DNA samples were dissolved in distilled H20 and used for real-time PCR (CFX96 Touch Real-Time PCR Detection System, Bio-Rad). Input and immunoprecipitated DNAs were PCR amplified in triplicate in SensiFAST™ SYBR No-Rox mix (Bioline).
10
FGFR3 Mutant Protein Analysis in HEK293 Cells
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HEK293 cells at 70%–80% confluence were transiently transfected with FGFR3 human constructs (FGFR3-Y373C, FGFR3-G380R, FGFR3-K650E). Transfected cells were incubated with (-)-epicatechin overnight and then lysed in RIPA buffer (50 mmol·L−1 Tris-HCl pH 7.6, 150 mmol·L−1 NaCl, 0.5% NP40, and 0.25% sodium deoxycholate, supplemented with protease and phosphatase inhibitors; Roche). Proteins were immunoprecipitated by the addition of 3 μL of rabbit anti-FGFR3 (Sigma-Aldrich, F0425) per 500 μg of protein with protein A-agarose (Roche). The excised FGFR3 protein was eluted and then denatured at 95 °C for 10 min in NuPAGE LDS sample buffer (Life Technologies, NP0008) and 2.5% β-mercaptoethanol.
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