For the analysis of the interaction between the V and NP of NDV, HEK293T cells in 60 mm diameter dishes were transfected with pCDNA-KHA-NP and pCDNA-2 × Flag-V or truncated V at a dose of 2 µg. At 36 h post-transfection, the cells were lysed with 500 µL of ice-cold RIPA lysis buffer supplemented with PMSF (1:1000) for 40 min, and the clarified supernatant was obtained after centrifugation at 12,000 rpm for 10 min at 4 °C. A total of 40 μL of supernatant was obtained for use as input samples. Mouse anti-Flag or anti-HA mAbs (Cell Signaling Technology, Danvers, MA, USA) were added to the remaining supernatant, and the complexes were incubated at 4 °C overnight. Thirty microliters of ice-cold PBS-washed protein A/G beads (Smart-Life Sciences, Changzhou, China) were added to the complexes and incubated at 4 °C overnight. The beads were further washed 3 times with ice-cold PBS, and 40 μL of 1× SDS loading buffer was added to suspend the pellets. The samples were heated at 95 °C for 10 min and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for Western blot analysis.
Protein a g beads
Manufactured by Smart-Lifesciences
Sourced in China
Protein A/G beads are a type of affinity chromatography resin used for the purification of antibodies from biological samples. They contain a recombinant form of either Protein A or Protein G, which have a high affinity for the Fc region of antibodies, allowing for selective capture and isolation of immunoglobulins.
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9 protocols using protein a g beads
1
Investigating NP-V Interaction in NDV
2
ChIP-seq protocol for transcription factors
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ChIP-seq was performed as previously described (30 (link)). Briefly, cells were cross-linked with 1% paraformaldehyde for 10 min and were quenched with glycine for 5 min. After cell lysis, fixed chromatin was fragmented to 200- to 600-bp DNA size using a Diagenode Bioruptor Plus. DNA fragments were immunoprecipitated with indicated antibodies and Protein A/G beads (Smart-Lifesciences, #SA032100). Specific antibodies against MYCN, MAX, AFF1, and AFF4 were generated by immunization in rabbits with recombinant antigens. One to 10 ng of immunoprecipitated DNA was used for DNA library preparation. ChIP-seq raw reads were aligned to the human genome (hg38) with Bowtie (version 1.1.2) using default settings. Read coverage was performed with deepTools and visualized with the UCSC genome browser. Peaks were called using MACS2 (version 2.1.2) with default parameters and a P value cutoff at 1 × 10−5 before annotation by ChIPseeker (version 1.28.3). Heatmaps and metagene plots were generated using the computeMatrix command from deepTools and NGStools, respectively.
3
Identifying ALKBH5-Associated Proteins
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To identify the proteins associated with ALKBH5, affinity purification was performed followed by MS analysis as described (38 (link)). Briefly, the nuclear extracts (100 mg) from the 293FT expressing HA-ALKBH5 were prepared as described previously. After the nuclear extracts were incubated with anti-HA-antibody overnight at 4 °C with rotation, 30 μl protein A/G beads (Smart-Lifesciences, SM01501) were added and incubated for 3 h. The beads were collected by centrifugation at 500g for 5 min and washed three times with cell lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.2 mM EDTA, 0.3% NP-40, 0.2 mM PMSF). The beads were then boiled in SDS loading buffer and run shortly into a SDS gel. A gel slice containing the purified proteins was isolated for MS analysis. The extracted tandem mass spectrometry data were processed using Mascot search engine. Tandem mass spectra were searched against SwissProt-Mouse database concatenated with reverse decoy database. Proteins containing unique mapped peptide with ion score meeting significant p value were considered.
4
Chromatin Immunoprecipitation Sequencing Protocol
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Chromatin immunoprecipitation sequencing was performed with 1 × 107 cells as previously described (Liang et al. 2015 (link)). The sheared chromatin was immunoprecipitated with 10 µg individual antibodies and 15 µL preblocked Protein A/G beads (Smart-Lifesciences). Library was prepared using the NEBNext ultra II DNA library prep kit for Illumina. For ChIP-Rx experiments, sonicated human chromatin was spiked-in with 10%–30% mouse chromatin to quantitatively normalize across experiments. Experimental details and sequencing data analysis are included in Supplemental Methods .
5
ChIP-Rx Protocol for Genome-wide Histone Profiling
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ChIP-Rx was performed with 1×107 human cells and 1×106 MEF cells for spike-in normalization as described in a previous study [21 (link)]. For immunoprecipitation, sonicated chromatin was incubated with 10 μg of specific antibodies and 15 μL of pre-blocked Protein A/G beads (Smart-Lifesciences). After extensive washes, the captured DNA was eluted for library preparation using the NEBNext Ultra II DNA library prep kit for Illumina before sequencing on a NovaSeq 6000. ChIP-Rx reads were aligned to the human genome (UCSC hg38) and mouse genome (mm10) with Bowtie2 version 2.2.6 [79 (link)], using parameters: --local --very-sensitive. The resulting reads were normalized with the aligned mouse reads. The aligned human BAM files were normalized and converted to bigwig files for visualization in the UCSC Genome Browser. Peaks were called using MACS2 version 2.1.2 with default parameters [81 (link)]. MEME-ChIP [82 (link)] was used to further analyze peak region motifs. Heatmap and metaplots were made for the indicated windows using the average coverage (r.p.m.).
6
Chromatin Immunoprecipitation Analysis of Histone Acetylation
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ChIP assay was carried out as the method of Lan et al. [26 (link)]. M1 macrophages were crosslinked with 1% formaldehyde for 10 mins and then stopped by 125 mM glycine. After lysis and sonication, 3 μg of Kla (PTM bio) antibody was incubated with chromatin sample overnight at 4°C, and 20 μl protein A/G beads (Smart lifesciences SA032005) were used for immunoprecipitation. Finally, DNA were purified with PCR recovery kit (QIAGEN #28006) and used for qPCR analysis. And the protein sample was analyzed by western blot to detect the histone acetylation level.
7
MYCN-ER Chromatin Immunoprecipitation Assay
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SHEP MYCN-ER cells were fixed with 1% formaldehyde diluted in PBS (Phosphate buffer saline) for 10 min at room temperature, quenched with 0.125 M glycine for 5 min at 37 °C. Fixed cells were lysed with SDS Lysis Buffer, followed by sonication (Bioruptor Pico Sonifier, Diagenode) to shear chromatin DNA to a size range of 500–1000 bp. Pre-cleared chromatin was immunoprecipitated with antibody against N-MYC overnight at 4 °C. Antibody-chromatin complexes were pulled down by pre-blocked Protein A/G beads (Smart-Lifesciences) for 1 h at 4 °C. De-crosslinked DNA was subjected to qPCR analysis using specific primers listed in Supplementary Table 2 .
8
Co-Immunoprecipitation and Western Blot Analysis
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Co-IP and Western blot analysis were performed as described previously [16 (link)]. Briefly, HEK293T cells were seeded to reach 60~70% confluence in multi-well plates or dishes, and transfected with plasmids for 36 h. Cells were washed with PBS and lysed in lysis buffer containing 50 mM Tris HCl, pH 7.6, 150 mM NaCl, 0.5% NP40, 1 mM EDTA, and protease inhibitor cocktail (APExBIO) at 4 °C for 10 min. Cell lysates were centrifuged twice at 14,000 rpm, 4 °C for 5 min, and supernatant was collected and incubated with an antibody overnight at 4 °C. The next day, 20 μL of protein A/G beads (Smart Lifesciences, Changzhou, China) was added to the supernatant, and the mixture was incubated for 1 h at 4 °C. After washing three times with lysis buffer, the beads were boiled in SDS loading buffer for 5 min, and the supernatant was used for Western blot. The following antibodies were used: Flag (Sigma-Aldrich, St. Louis, MO, USA, #F1804 and #F7425) and Myc (Sigma-Aldrich, St. Louis, MO, USA #M5546).
9
Vinculin-Rictor Interaction Analysis
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To investigate the interaction of vinculin and Rictor, immunoprecipitation was performed. Wild‐type cells cultured on the three surfaces were collected and lysed on ice for 30 minutes, sonicated and centrifugated, and 3% supernatant was collected as input. Vinculin antibody (CST) was added to the incubation at 4℃ overnight, followed by 3‐hour incubation with Protein A/G beads (Smart‐Lifesciences). Immunoprecipitates were washed three times and resuspended when buffer is loaded for SDS‐PAGE analysis.
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