RPPA was performed at MDACC Core Facility using all antibodies available in the core. RPPA methodology was described previously (42). For multiple markers, Western blotting was conducted according to previously published procedures.38 The same amounts of cell lysates were loaded in each membrane for each target. actin was used as a loading control. The antibodies used for Western blotting were BCL-2 (Agilent DAKO), MCL-1 (Santa Cruz Biotechnology), BCL-XL, BAX, BAK, PUMA (Cell Signaling Technology), BCL-2 A1, NOXA, BIM (Abcam), actin, and tubulin (Sigma). The antibodies used for co-IP were BIM, mouse immunoglobulin G (Santa Cruz Biotechnology), and MCL-1 (Millipore Sigma). Co-IP experiments were performed using a Dynabeads Protein A Immunoprecipitation Kit (ThermoFisher) following the manufacturer’s instructions. In brief, cells were washed with cold phosphate buffered saline and lysed with cell lysis buffer (Cell Signaling Technology), then the cleared lysates were incubated with antibody-conjugated Dynabeads. The bead-protein complexes were then isolated using a magnet and washed. The proteins were later eluted from the beads and used for subsequent experiments.
Dynabeads protein a immunoprecipitation kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Dynabeads Protein A Immunoprecipitation Kit is a versatile tool designed for the rapid and efficient purification of target proteins from complex biological samples. The kit utilizes magnetic Dynabeads coated with Protein A, which selectively binds to the Fc region of antibodies. This allows for the capture and isolation of protein complexes, enabling researchers to study protein-protein interactions and post-translational modifications.
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Lab products found in correlation
Protease inhibitor cocktail tablet, by Roche (4 mentions)
Mouse anti human myd88 antibody, by Santa Cruz Biotechnology (2 mentions)
Antimouse igg horseradish peroxidase hrp antibody, by Cell Signaling Technology (2 mentions)
Np40 cell lysis buffer, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Nupage 3 8 tris acetate gel, by Thermo Fisher Scientific (2 mentions)
α wdfy3 antibody, by Novus Biologicals (2 mentions)
α wdfy3primary antibody, by Novus Biologicals (2 mentions)
Ir secondary antibody, by LI COR (2 mentions)
Odyssey ir scanner, by LI COR (2 mentions)
Odyssey blocking buffer, by LI COR (2 mentions)
Immobilon pvdf membrane, by Merck Group (2 mentions)
Immobilon western chemiluminescent hrp substrate detection reagent, by Merck Group (2 mentions)
Nanodrop lite, by Thermo Fisher Scientific (2 mentions)
Biotin switch assay kit, by Abcam (2 mentions)
Tubulin, by Merck Group (1 mentions)
Bcl 2, by Agilent Technologies (1 mentions)
Mcl 1, by Santa Cruz Biotechnology (1 mentions)
Bcl 2 a1, by Abcam (1 mentions)
Mouse immunoglobulin g, by Santa Cruz Biotechnology (1 mentions)
51 protocols using dynabeads protein a immunoprecipitation kit
1
Comprehensive Protein Analysis of Apoptosis
2
Immunoprecipitation of Secreted NMU
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Cells were cultured on 75 cm2 flasks to 80–90 % confluency, next washed (PBS) and the medium was changed to 5 ml of fresh growth medium without FBS. After 48 h, 2 ml of conditioned medium (CM) were collected, centrifuged (1000 x g, 4 °C, 20 min) and immediately used for NMU immunoprecipitation performed with Dynabeads™ Protein A Immunoprecipitation Kit (Thermo Fisher Scientific) according to manufacturer’s instruction. 20 µl of Dynabeads were conjugated with 2 µg of rabbit anti-NMU antibodies (Abbexa, Cambridge, GB) or IgG antibodies from rabbit serum (Sigma-Aldrich) as a control. Antibodies-antigen complexes were eluted with SDS sample buffer with β-mercaptoethanol at 95 °C for 5 min and analysed by immunoblotting.
A part of the gel with HCT116 CM immunoprecipitation was fixed in methanol. The excised gel slice, corresponding to the NMU signal on the Western blot was send for the mass spectrometry identification (described in Additional File1 ; commercial service at the Institute of Biochemistry and Biophysics of the Polish Academy of Sciences, Warsaw, Poland).
A part of the gel with HCT116 CM immunoprecipitation was fixed in methanol. The excised gel slice, corresponding to the NMU signal on the Western blot was send for the mass spectrometry identification (described in Additional File
3
TLR9-MyD88 Interaction Modulation
4
Immunoblotting and Immunoprecipitation Assay Protocol
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Immunoblotting and immunofluorescence staining were carried out as previously described (Jing et al., 2011 ). Immunoprecipitation was performed using Dynabeads Protein A Immunoprecipitation Kit according to the manufacturer's protocol (#10006D, Thermofisher, USA). Antibodies used in this study: HDAC4 (#7628, Cell signaling, Germany), FoxO1 (#2880, Cell signaling, Germany), Pdx1 (sc‐14664, Santa Cruz, USA), acetylated lysine (#9441, Cell signaling Germany), α‐Tubulin (#T6199, Sigma, USA), and total histone H3 (#4499, Cell signaling, Germany) (Table S4 ).
We used GraphPad Prism 5.0 software (GraphPad Software). Differences between three or more groups were assessed by 1‐way ANOVA with Dunnett's correction. p < 0.05 were considered significant.
We used GraphPad Prism 5.0 software (GraphPad Software). Differences between three or more groups were assessed by 1‐way ANOVA with Dunnett's correction. p < 0.05 were considered significant.
5
Zinc Content Measurement in PARP-1 Protein
6
ChIP-seq and co-IP Analysis of miR-577 Regulation
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For the ChIP assay: After cells were cross-linked and lysed, DNA fragments were sonicated on ice to lengths between 200 and 1,000 bp. Anti-p65 antibody (catalog no. 26156, 1:50, Cell Signaling Technology) and control immunoglobulin G were used to precipitate protein-DNA complexes. The detail processes were performed according to the instructions of the Pierce Agarose ChIP Kit (Thermo Scientific, Waltham, MA, USA; catalog no. 26156). Finally, PCR was done to examine the putative p65-binding sites in the miR-577 promoter with its specific primers (Table S5 ). For the co-immunoprecipitation (coIP) assay: About 80%–90% adherent MGC803 and MKN45 cells were extracted by cell lysis buffer for IP. Then coIP assays were performed with the Dynabeads Protein A Immunoprecipitation Kit (Thermo Scientific, Waltham, MA, USA; catalog no. 10006D) according to the manufacturer’s protocols. Immunoprecipitated proteins (SDPR and ERK) were proved by western blot.
7
Ebi3 Immunoprecipitation from Exosomes
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The i35-Exosome lysates were incubated with antibodies (4 μg of anti-Ebi3 or Normal IgG) overnight at 4°C. Next day, magnetic beads from Dynabeads Protein A Immunoprecipitation Kit (Thermo Fisher Scientific (Waltham, MA) were incubated with lysates for 1 h at 4°C and precipitated beads was washed and proteins were eluted and boiled for 10 min at 95°C. Samples were fractionated on 4–12% gradient SDS-PAGE and incubated with Ebi3 or p35 antibodies. After secondary antibodies reaction, signals were detected with LI-COR system. Image studio software was used for data analysis.
8
Immunoprecipitation of SARS-CoV-2 ORF7a
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HEK 293T cells were seeded in a 10 cm culture dish (3.5 × 106 cells) and transfected with pCDNA SARS-CoV-2 ORF7a-V5/His (WT/H47Y; 7 µg) or empty vector plasmids. At 48 hours posttransfection, cells were washed in cold PBS and lysed in 700 µL of NP-40 lysis buffer containing 5% glycerol and 1× Halt Protease and phosphatase inhibitors (Thermo Fischer Scientific) for 30 min at 4 °C. Lysates were clarified by centrifugation and used for immunoprecipitation using the Dynabeads protein A immunoprecipitation kit (Thermo Fisher Scientific). Dynabeads (30 μL) were preincubated with either mouse anti-V5 (1:200) (Thermo Fisher Scientific, R960-25) or rabbit anti-HLA class I ABC (1: 200) (Proteintech, 15240-1-AP, Rosemont, IL, USA) antibodies for 20 min at RT. Cell lysates (2000 μg) were incubated with antibody-coated Dynabeads followed by overnight incubation at 4 °C. The next day, Dynabeads were then washed and eluted. The eluted fractions and 10% clarified lysates (inputs) were subjected to Western blot analysis (see Immunoblotting section).
9
Ubiquitination Assay for SARS-CoV-2 ORF7a
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The ubiquitination assay was performed as before with some modifications [15 (link)]. Briefly, HEK 293T cells (0.5 × 106 cells/well) were seeded in duplicate in a 6-well plate. The next day, cells were transfected with pCDNA SARS-CoV-2 ORF7a-V5/His (WT/H47Y; 5 µg) and HA-ubiquitin (500 ng) or empty vector plasmids. At 48 hours posttransfection, cells were washed with cold PBS and lysed in 1× RIPA lysis buffer (300 µL). Clarified lysates (2000 µg) were used for immunoprecipitation using the Dynabeads protein A immunoprecipitation kit (Thermo Fisher Scientific) as per the manufacturer’s instructions. Briefly, 50 µL of beads was preincubated with anti-V5 antibody (1:200) (Thermo Fisher Scientific, R960-25) for 20 min at RT, then washed and incubated with cell lysates overnight at 4 °C. The next day, the beads were washed, eluted, and subjected to immunoblot analyses (see Immunoblotting section). For inputs, 10% of clarified lysates were resolved.
10
Isolation and Co-Immunoprecipitation of GATA-1, FOG-1, and PU.1
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GATA-1 or PU.1 protein was isolated by immunoprecipitation and GATA-1/FOG-1 complex was co-immunoprecipitated as previously described45 (link). Briefly, GATA-1, FOG-1, or PU.1 were purified using GATA-1 (D52H6) XP rabbit monoclonal antibody, PU.1 (9G7) rabbit monoclonal antibody (Cell Signaling Technologies), or FOG-1 rabbit polyclonal antibody (ab86281; Abcam), respectively using Dynabeads Protein-A Immunoprecipitation Kit (ThermoFisher Scientific). Immunoprecipitated GATA-1, FOG-1, and PU.1 were eluted from the beads and utilized for downstream experiments. GATA-1 and FOG-1 interaction was determined in co-immunoprecipitated GATA-1/FOG-1 complex by western blotting. Densitometry summaries were obtained from 3 independent blots after normalization to the density level of the whole image. To prepare samples for ICP-MS, a non-denaturing method was used to elute GATA-1 or PU.1 from beads, and the solution adjusted to pH > 7 using the neutralizing buffer provided in the immunoprecipitation kit.
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