RASFs were lysed in RIPA at 4 °C for 30 min, the lysates were centrifuged at 14,000 × g for 15 min, and the supernatants were collected. A small amount of whole-cell lysate was retained as the input. Next, the supernatant samples were incubated with the FoxC1 antibody (ab5079), β-catenin antibody (L87A12) (Cell Signaling) or normal IgG antibody (Cell Signaling) combined with protein A/G magnetic beads (Thermo Scientific) on a rotating device at 4 °C overnight. The bead-complexes were collected by centrifugation at 14,000 × g for 1 min at 4 °C. Finally, the beads were washed with lysate, and the protein was boiled with 10% SDS and subjected to immunoblot analyses.
Normal igg antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Normal IgG antibody is a type of immunoglobulin G (IgG) that does not have specific antigen-binding activity. It serves as a control for experiments that use IgG antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Protein a g magnetic bead, by Thermo Fisher Scientific (1 mentions)
Chip assay kit, by Cell Signaling Technology (1 mentions)
Phf6 antibody, by Merck Group (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Magnetic separator, by Merck Group (1 mentions)
Magna rip kit, by Merck Group (1 mentions)
Syber green master mix, by Roche (1 mentions)
Mircury rna isolation kit, by Qiagen (1 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (1 mentions)
Imprint rna immunoprecipitation kit, by Merck Group (1 mentions)
4 protocols using normal igg antibody
1
Immunoprecipitation of FoxC1 and β-catenin
2
RNA-Seq and ChIP-Seq of Phf6 and Jak3 in K562 cells
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For RNA-Seq analysis of Phf6 WT/KO + JAK3M511I cells, 1 µg RNA per sample was used for RNA sample preparations. Transcriptome sequencing was performed on Illumina NovaSeq 6000 platform (Illumina, CA, USA) to a total target depth of 10 million 150 bp paired end reads. Differential expression analysis was performed by DESeq2 R package (1.16.1).
The ChIP assay was performed with the ChIP assay kit (Cell Signaling Technology, Boston, MA, USA) according to manufacturer’s recommendations. Chromatin from cross-linked HA-PHF6-overexpressed K562 or control cells was sheared using an ultrasonicator (Covaris, S220, ABI, New York, USA) to obtain DNA (100–400 bp). Immunoprecipitation was conducted with ChIP-grade HA-tag antibody (Abcam, ab9110, Cambridge, UK), PHF6 antibody (Sigma-Aldrich, HPA001023, Missouri, USA) or normal IgG antibody (Cell Signaling Technology, CST2729, Boston, MA, USA).
RNA-Seq and ChIP-Seq data are available at GEO under accession number GSE159444 and GSE159549.
The ChIP assay was performed with the ChIP assay kit (Cell Signaling Technology, Boston, MA, USA) according to manufacturer’s recommendations. Chromatin from cross-linked HA-PHF6-overexpressed K562 or control cells was sheared using an ultrasonicator (Covaris, S220, ABI, New York, USA) to obtain DNA (100–400 bp). Immunoprecipitation was conducted with ChIP-grade HA-tag antibody (Abcam, ab9110, Cambridge, UK), PHF6 antibody (Sigma-Aldrich, HPA001023, Missouri, USA) or normal IgG antibody (Cell Signaling Technology, CST2729, Boston, MA, USA).
RNA-Seq and ChIP-Seq data are available at GEO under accession number GSE159444 and GSE159549.
3
Argonaute 2 RNA Immunoprecipitation
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The RIP assay was performed using a Magna RIP kit (Millipore, Billerica, MA, USA). The mitochondrial fraction was lysed with RIP lysis buffer. After centrifugation, the supernatant was incubated with an anti-Argonaute 2 (anti-Ago2) antibody (Abcam, Cambridge, MA, USA) or normal IgG antibody (Cell Signaling Technology, Billerica, MA, USA) overnight at 4°C. Then, A/G magnetic beads were added to the samples and incubated at 4°C for 4 h. The beads were harvested using a magnetic separator (Millipore) and used to isolate RNA with PCA reagent (phenol:chloroform:isoamyl alcohol at a ratio of 125:24:1). The isolated RNA was subjected to qRT-PCR analysis.
4
RNA Immunoprecipitation and Quantification
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The RNA immunoprecipitation was performed according to the manufacture’s manual (Imprint® RNA Immunoprecipitation Kit; Sigma-Aldrich, St. Louis, MO). Briefly, control or Sig-1R KO HEK cells were harvested in lysis buffer containing protease inhibitor cocktail and ribonuclease inhibitor, and incubated on ice for 15 min. After centrifugation, the lysate was incubated with eIF4E antibody (15 μg; MBL, RN001P) or normal IgG antibody (15 μg; Cell Signaling) overnight at 4 °C. Samples were then incubated with magnetic beads (DynaBeads Protein A, Invitrogen by ThermoFisher Scientific, Waltham, MA) at 4 °C for 2 h for immunoprecipitation. Samples were washed 5 times with washing buffer. The RNA was then purified by miRCURYTM RNA isolation kit (EXIQON, product #300110; Woburn, MA). A reverse transcription reaction (RNA to cDNA EcoDryTM Premix kit; Clontech Laboratories, Cat#639543; Mountain View, CA) was performed to synthesize the complementary DNA and quantitative PCR was conducted using Syber Green Master Mix (Roche) and specific primers to quantify the cDNA level.
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