Cells were fixed with 1% formalin and sonicated. Then sheared DNA was incubated with antibodies against MYCN (Santa Cruz Biotechnology, sc-53993) or IgG control (Santa Cruz Biotechnology, USA, sc-2027). DNA-protein-antibody complexes were incubated with Protein A Agarose/Salmon Sperm DNA Beads (Merck, Germany, CS204457). The beads were washed sequentially with gradient salt buffer and eluted in 1% SDS/NaHCO3. Finally, ChIP DNA was analyzed by qPCR.
Salmon sperm dna protein a agarose beads
Manufactured by Merck Group
Sourced in United States
Salmon sperm DNA/protein A agarose beads are a type of affinity chromatography resin. They are composed of agarose beads with covalently attached salmon sperm DNA and protein A. These beads are commonly used for the purification of antibodies and other biomolecules from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti ha antibody, by Abmart (3 mentions)
Control igg, by Santa Cruz Biotechnology (2 mentions)
Misonix sonicator 3000, by Bioventus (2 mentions)
Millipore chip kit, by Merck Group (2 mentions)
Radioimmunoprecipitation assay buffer, by Merck Group (2 mentions)
Bioruptor sonicator, by Diagenode (2 mentions)
Sc 53993, by Santa Cruz Biotechnology (1 mentions)
Pcr purification kit, by Qiagen (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Genome analyzer iix, by Illumina (1 mentions)
Proteinase k, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Nf κb, by Cell Signaling Technology (1 mentions)
Ab7970, by Abcam (1 mentions)
Anti ctcf antibody, by Cell Signaling Technology (1 mentions)
Bradford colorimetric assay, by Bio-Rad (1 mentions)
Phosphostop, by Merck Group (1 mentions)
Cebpb, by Santa Cruz Biotechnology (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
24 protocols using salmon sperm dna protein a agarose beads
1
MYCN Chromatin Immunoprecipitation Protocol
2
Regulation of MMP2 and MMP9 Transcription
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We also investigated whether EGF-dependent NAB2 upregulation results in altered transcriptional regulatory efficiency for Sp1 and EGR1. For this, we performed ChIP analysis with an Sp1 or EGR1 antibody using MMP2 and MMP9 genes in FaDU cells with control siRNA or siNAB2 pretreatment. In addition, Sp1 ChIP analysis was performed in EGR1- and/or NAB2-overexpressing cells. After 48 h, cross-linking was performed by formaldehyde. Cell pellets were resuspended in SDS lysis buffer and sonicated for chromatin fragmentation to an average length of less than 500 bp. ChIP was performed overnight at 4 °C with anti-Sp1/anti-EGR1 antibody or normal rabbit IgG. Next, 50 μL of protein A agarose/salmon sperm DNA beads (Merck Millipore, Billerica, MA, USA) was added and incubated for 4 h. Reverse crosslinking was carried out by incubating the samples overnight with 0.3 M NaCl at 65 °C. RNA and unbound protein were removed by adding RNase A and proteinase K, respectively. DNA was extracted using the PCR purification kit (Qiagen, Valencia, CA, USA) and suspended in 50 μL of Tris-ethylenediaminetetraacetic acid buffer. qPCR was performed using the immunoprecipitated chromatin or input chromatin with primers flanking the EGR1 binding sequences of each promoter (MMP2-1, −4377 to −4363; MMP2-2, −1489 to −1484; MMP2-3, −411 to −406; MMP9-1, −6106 to −6097; MMP9-2, −1746 to −1737).
3
ChIP-seq analysis of transcription factors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HCC cells were fixed in 3.7% formaldehyde for DNA cross-linking and lysed with SDS buffer, followed by sonication. Sheared DNA fragments were then incubated with antibodies against MYC, MXI1, HIF-1β, or immunoglobulin G (IgG) control (table S2) together with Protein A Agarose/Salmon Sperm DNA Beads (Merck Millipore, Burlington, MA, USA). After the incubation step, DNA-protein-antibody complexes were washed with salt buffer with gradient concentrations and then eluted in 1% SDS/NaHCO3. ChIP DNA samples were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) using primers flanking the putative E-box element or HRE (table S3).
4
ChIP Assay for HNF-4α Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ChIP assay was performed based on previous reported protocols 44 . Hepa1-6 cells were co-transfected with plasmids encoding HNF-4α and WT or Mutant PRMT1. After preclearing the chromatin with protein A-agarose/salmon sperm DNA beads (16-157, Sigma-Aldrich, MO, USA) with rotation for 2 hours at 4 °C, samples were divided equally and incubated with 5 μg of either anti-HNF-4α antibody or non-immune Rabbit IgG (in-house production) with rotation for overnight at 4 °C. DNA was washed and eluted with 30 μl of H2O for further analysis by qPCR.
5
ChIP-qPCR Protocol for Protein-DNA Interactions
6
Telomere Length Quantification in Arabidopsis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Approximately 4–6 grams of Arabidopsis seven day-old seedlings were used for each genotype. The protocol was adapted from [74] (link) with minor changes. Sonication was performed on ice after crosslinking and nuclei extraction using (Fisher Scientific) with 4 cycles of 15 sec on and 1 min off per sample at 40% amplification. Immunoprecipitation (IP) was performed using rabbit anti-TERT antibody and Protein-A agarose/salmon sperm DNA beads (Millipore). Eluted DNA was subjected to Southern dot blotting using a telomeric [32P] 5′ end-labeled oligonucleotide probe. Stripping and rDNA hybridization performed as previously described using a combination of 5S (5′-TTGCAGAATCCCGTGAACCATCGAGT-3′ ) and 18S (5′-TGGAGCCTGCGGCTTAATTTGACTCA-3′ ) rDNA oligo-probes [59] (link). Quantification was performed on at least three independent biological replicates using Quantity One software (Bio-Rad).
7
Characterization of ARF8-DNA Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EMSA and ChIP analyses were conducted by using previously described methods [47 (link)]. For EMSA, the ARF8 protein was fused to a His tag for six tandem histidine residues, and a fusion protein was produced by prokaryotic expression [48 (link)]. Previously designed WT probe, 5'-CATTATTTACGTGTCT GTTTTCCTG-3', and mutant 5'-CATTATTTACGtagat GTTTTCCTG -3' [26 (link)] were used in the EMSA after labeling with biotin. The chromatin used in the ChIP assay was isolated from the top sixth leaves of 50-day-old plants as described elsewhere [50 (link)]. Complexes of chromatin fragments and NtARF8-YFP (ARF8-IP complexes) were precipitated with a specific YFP antibody (Santa Cruz) and the Protein A agarose/salmon sperm DNA beads (Millipore). The NtARF8-IP complexes bound to the beads were collected by centrifugation. Concentrations of the antibody and chromatin were 10 μg and 50 nM per immunoprecipitation, respectively. Similar precipitation and centrifuge procedures were conducted in the absence of the antibody in the control. Chromatin without precipitation were used as input. DNA samples from ChIP, control, and input were analyzed separately by PCR using primers specific to the promoter and CDS of the GH3 gene (Additional file 9 : Table S2). The PCR products were confirmed by sequencing.
8
ChIP-seq protocol for chromatin analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed with 1% formaldehyde for 10 min at 37 °C, lysed in SDS lysis buffer (50 mM Tris pH8, 10 mM EDTA, 1% SDS, protease inhibitor cocktail (Roche)) and sonicated. Supernatant was diluted 10 times in IP dilution buffer (16.7 mM Tris pH8, 167 mM NaCl, 1.2 mM EDTA, 1.1% TritonX-100, 0.01% SDS, protease inhibitor cocktail (Roche)) and immunoprecipitations were carried out overnight with specific antibodies. Immunoprecipitated chromatin was collected using Protein A Agarose/Salmon Sperm DNA beads (Millipore) and, after washing and elution, reverse cross-linking was carried out with 0.2 M NaCl at 65 °C overnight. The chromatin was then digested by 20 μg of Proteinase K (Invitrogen) for 1 h at 45 °C and isolated by phenol–chloroform extraction. PCR reactions were performed using SYBR Green PCR Master Mix (Applied Biosystems) and specific primers sequences are listed below.
For sequencing, 10 ng of purified DNA from ChIP was adapter-ligated, PCR amplified and sequenced on the Illumina Genome Analyzer IIx as single-end 50 base reads following Illumina's instructions. Sequence reads were mapped to reference genome mm9/NCBI37 using Bowtie v0.12.7.
For sequencing, 10 ng of purified DNA from ChIP was adapter-ligated, PCR amplified and sequenced on the Illumina Genome Analyzer IIx as single-end 50 base reads following Illumina's instructions. Sequence reads were mapped to reference genome mm9/NCBI37 using Bowtie v0.12.7.
9
ChIP Assay for Transcription Factors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The ChIP analysis was performed using the Millipore ChIP kit (Millipore, Billerica, MA, USA), following the manufacturer’s instructions with minor modifications. For each assay, the THP-1 monocytes were inoculated into a 10-cm dish (a total of 5 × 106 cells) and fixed with 1% formaldehyde. Cell pellets were resuspended in SDS lysis buffer containing protease inhibitors (1 mM PMSF, 1 µg/mL aprotinin, and 1 µg/mL pepstatin A). The samples were sonicated with a Misonix sonicator 3000 (Misonix, Farmingdale, NY, USA), centrifuged, and diluted 10-fold in ChIP dilution buffer. After removing an aliquot (whole-cell extract input), the chromatin samples were incubated at 4 °C overnight with antibodies against AP-1 (#9165, Cell Signaling) or NF-κB (#8242, Cell Signaling). The samples were then precipitated by binding to protein A-agarose/salmon sperm DNA beads (Millipore, Billerica, MA, USA). The immunoprecipitated chromatin was analyzed by PCR using primers for the Mac-1 gene promoter. Cycling parameters were 58 °C for 1 min and 95 °C for 30 s, followed by 40 cycles.
10
Chromatin Immunoprecipitation (ChIP) Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed and proteins were cross-linked to DNA using 0.75% paraformaldehyde (PFA) in PBS for 30 min. Glycine (125 mM) was added to quench cross-linking reaction, and cells were collected and lysed in radio-immunoprecipitation assay buffer (Sigma-Aldrich) containing 1× protease and phosphatase inhibitor cocktail, and DNA was fragmented by sonication to 500–1000-bp size. Protein A-agarose/salmon sperm DNA beads (Millipore) were incubated with DNA and antibody against the protein of interest or IgG, and the samples were immunoprecipitated overnight with rotation at 4 °C. DNA was eluted by adding elution buffer (10 mM EDTA, 1% SDS, 50 mM Tris-HCl, pH 8.0). Input samples were incubated with RNase A (20 μg/mL) and Proteinase K (0.5 mg/mL) at 65 °C overnight to reverse cross-linking. DNA was precipitated with isopropanol, and qRT-PCR was performed to assess the binding of the protein to the anticipated binding sites in DNA using the primers used listed in Supplementary Table 1 .
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!