Splenic B-1a cells from VH12/Vκ4 transgenic Bhlhe41Tag/Tag mice were enriched by magnetic depletion of cell of other lineages. To this end, splenocytes were stained with a cocktail of PE-labeled antibodies against CD23, CD4, CD8, TCRβ, TCRγδ, NK1.1, CD11c, CD11b, Gr-1 and Ter119 followed by magnetic depletion with anti-PE MicroBeads (Miltenyi Biotec). Chromatin from 6 × 107 B-1a cells was prepared using a lysis buffer containing 0.25% SDS. The cells were subjected to ChIP as described56 (link) with minor modifications. Anti-V5 agarose beads (Sigma-Aldrich) were used for precipitation. Sequencing libraries were prepared with the NEBNext Ultra Ligation Module and NEBNext End Repair/dA-tailing module followed by PCR amplification with the KAPA Real Time Amplification kit (KAPA Biosystems) and Illumina deep sequencing.
Anti v5 agarose beads
Manufactured by Merck Group
Sourced in United States
Anti-V5 agarose beads are a laboratory product used for protein purification. They are composed of agarose beads coated with antibodies specific to the V5 epitope tag. The primary function of these beads is to facilitate the capture and isolation of V5-tagged proteins from complex biological samples.
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Lab products found in correlation
Bioruptor, by Diagenode (6 mentions)
Mini beadbeater, by Biospec (5 mentions)
Sybr green mix, by Thermo Fisher Scientific (4 mentions)
Anti flag m2 affinity gel, by Merck Group (3 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (3 mentions)
Kapa real time amplification kit, by Roche (2 mentions)
Anti pe microbeads, by Miltenyi Biotec (2 mentions)
Ubiquitin activating enzyme e1, by R&D Systems (2 mentions)
E2 ubiquitin conjugating enzyme ubch7, by R&D Systems (2 mentions)
Ubiquitin, by Merck Group (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Anti ha agarose beads, by Merck Group (2 mentions)
Protein a magnetic beads, by Thermo Fisher Scientific (2 mentions)
Flag or v5 antibody conjugated beads, by Merck Group (2 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Fugene 6 transfection reagent, by Promega (1 mentions)
M2 anti flag agarose beads, by Merck Group (1 mentions)
Gfp trap agarose beads, by Proteintech (1 mentions)
Protein a sepharose, by GE Healthcare (1 mentions)
22 protocols using anti v5 agarose beads
1
Enrichment and ChIP-seq of Splenic B-1a Cells
2
Immunoaffinity Purification of V5-tagged Proteins
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Exponentially growing cells were harvested and the pellet was resuspended in an equal volume of lysing buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol and 2 mM MgCl2) supplemented with 0.1% NP-40 and protease and phosphatase inhibitors [50 mM NaF, 5 mM Na4P2O7, 0.1 mM Na3VO4, 1 mM β-glycerophosphate, 1 mM PMSF and Roche cOmplete Mini Protease tablets (one tablet per 10 ml of buffer)]. The suspension was frozen in liquid nitrogen and ground into a powder with a SPEX SamplePrep 6870 freezer mill. 1 ml of ice-cold lysing buffer (plus protease inhibitors) was added to 1 g of the frozen powder and gently thawed on ice. 375 units of benzonase (Novagen, 250 U/µl; Merck) was added, and the mixture was rotated at 4°C for 1 h. The lysate was then clarified by spinning at 21,130 g at 4°C. This was followed by measurement of protein concentration in the clear lysate with a Bradford protein assay (Biorad). Lysate containing 10 mg of total protein was added to 50 µl of packed anti-V5 agarose beads that were prepared according to manufacturer's instructions (Sigma-Aldrich) and incubated overnight at 4°C. Next day, the sample was spun at 5000 g for 30 s and washed five times with lysing buffer (plus protease inhibitors). Bound proteins were eluted twice with 50 µl 4× SDS loading buffer. The input and eluates were analyzed by western blotting as described below.
3
Substrate SUMOylation Assay Protocol
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Candidate substrates were subcloned into the PCAGIG-V5 mammalian expression vector, SUMO1 and SUMO2 were subcloned into PCAGIG-MYC mammalian expression vector, and E3 ligases were subcloned into PSG5-FLAG mammalian expression vector. V5-substrate and MYC-SUMO were transfected together with and without FLAG-E3 ligases constructs with Fugene 6 transfection reagent (Promega) into HeLa cells seeded at 2E105 cells per well. After 48 h, the cells were washed with phosphate buffered saline and lysed in RIPA buffer containing 20 mm N-ethylmaleimide and 1% SDS, to inhibit deSUMOylation and dissociate noncovalent protein complexes. Anti-V5-agarose beads were added (Sigma-Aldrich, St. Louis, MO) for 2 h to immunoprecipiate the substrate. Immunoprecipitates were resolved by SDS-PAGE and subject to immunoblotting to detect SUMOylation.
4
Detecting Ubiquitination of NEDD4L Protein
5
Immunoprecipitation and Western Blot Analysis
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Cells were lysed with RIPA lysis buffer supplemented with phosphatase inhibitors (1 mM Na3VO4, 2 mM NaF and 200 μM sodium pyrophosphate). Cell lysates containing equal amount of proteins were incubated with anti-Flag agarose beads (M2; Sigma-Aldrich), anti-V5 agarose beads (Sigma-Aldrich) or GFP-Trap agarose beads (Chromotek) at 4 °C for 2 h. Alternatively, cells were lysed with NP40 lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.5], 1% NP40, 1% sodium deoxycholate, 1 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mM PMSF) supplemented with phosphatase inhibitors. Cell lysates were pre-cleared with protein A Sepharose (GE Healthcare) at 4 °C for 1 h and incubated with various antibodies at 4 °C for overnight, followed by 2 h of incubation with protein A Sepharose at 4 °C. After washing the beads with lysis buffer for three times, proteins bound on beads were analyzed by western blot.
6
Co-immunoprecipitation of Exogenous and Endogenous Proteins
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For co-IPs of exogenous protein interactions, the injected embryos were lysed with TNSG buffer including protease inhibitors. Tagged proteins were precipitated with monoclonal anti-HA agarose beads (A2095; Sigma-Aldrich), anti-V5 agarose beads (A7345; Sigma-Aldrich), or anti-Flag M2 affinity gel (A2220; Sigma-Aldrich). For GFP-tagged protein pull-down, anti-GFP antibody was used. The resultant immunocomplexes were formed for 8 h at 4°C on a nutator and washed with lysis buffer four times. For co-IPs of endogenous protein interactions, HT29 and LS174T cells were lysed with TNSG containing 0.5% NP-40, and 10 µl of anti-Dvl2 antibody was incubated with 2 mg of total lysates at 4°C overnight, followed by an additional incubation with protein A/G plus agarose beads (sc-2003; Santa Cruz Biotechnology) for 2 h. Bead immunocomplexes were washed three times with the same lysis buffer. To examine coprecipitation of Drg1 with Dvl2, IB with Drg1 antibody was followed.
7
Chromatin Immunoprecipitation Protocol for Rme1 Binding
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Chromatin immunoprecipitation experiments were executed as described previously (Weidberg et al. 2016 (link)). Cells were fixed with 1 % formaldehyde for 20 min, the reaction was quenched with 125 mM glycine. Cells were disrupted using a mini beadbeater (BioSpec), and crosslinked chromatin was sheered by sonication using the Bioruptor (Diagenode, 7 cycles of 30 s on/off). Chromatin extracts were then incubated with anti-V5 agarose beads (Sigma) for 2 h at room temperature, and beads were washed accordingly. To measure Rme1 binding, input and ChIP samples were quantified by real-time PCR using SYBR green mix (Life Technologies) and primers corresponding to the IRT1 promoter on a 7500 Fast Real-Time PCR system (Life Technologies). The mating-type locus (HMR) was used as a non-binding negative control. Primer sequences are available on request.
8
Endogenous and Exogenous Protein Interactions
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The injected embryos were harvested at stages 11–12 and lysed with TNSG buffer containing protease inhibitors (cOmplete Protease inhibitor cocktail [Sigma-Aldrich] and PMSF) and phosphatase inhibitors (sodium orthovanadate and β-glycerophosphate). Exogenous tagged proteins were pulled down with anti-V5 agarose beads (A7345; Sigma-Aldrich), anti-Flag M2 affinity gel (A2220; Sigma-Aldrich), or HA epitope tag antibody agarose conjugate (sc-500777A; Santa Cruz Biotechnology) for 6–8 h at 4°C on a nutator, followed by washing with TNSG lysis buffer. For endogenous protein co-IPs, HEK293T cells were lysed with NP-40 lysis buffer (25 mM Hepes, 150 mM KCl, 1.5 mM MgCl2, 0.5% NP-40, 5% glycerol, and protease and phosphatase inhibitors). 1 mg of total lysates were incubated with 1 µg of Dyrk1a antibody (ab69811; Abcam) at 4°C overnight, followed by an additional incubation with protein A/G plus agarose beads (sc-2003; Santa Cruz Biotechnology) for 2 h and washing with the lysis buffer four times. Co-IPs were repeated at least three times.
9
Chromatin Immunoprecipitation of V5-Tagged Ume6 Transcription Factor
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Diploid cells suspended in SPO for 4 h were collected for chromatin immunoprecipitation (ChIP) experiments for the V5 tagged Ume6 transcription factor. Cells expressing untagged controls were included in the analysis. Cells were fixed in 1.0% w/v of formaldehyde for 15–20 min at room temperature and quenched with 100 mM glycine. After breaking cells using a mini beadbeater (BioSpec), crosslinked chromatin was sheared by sonication using Bioruptor (Diagenode, 6 cycles of 30 s on/off). Extracts were incubated with anti V5 agarose beads (Sigma) for 2 h, and beads were washed. Subsequently, reverse cross-linking was done in Tris-EDTA buffer (100 mM Tris pH 8.0, 10 mM EDTA, 1.0% v/v SDS) at 65 °C overnight. After 2 h of proteinase K treatment, DNA was purified on column (Macherey-Nagel). The concentration of purified DNA was quantified using the Qubit dsDNA HS assay kit (Q32851, Invitrogen). DNA was then used as input for the KAPA Hyper Prep Kit (KK8504, Roche) and ligated to KAPA single indexed adapters Set A (KK8701, Roche). Libraries were constructed according to the manufacturer’s instructions. Purified libraries were further quantified and inspected on a Tapestation (Agilent Technologies) and sequenced on an Illumina HiSeq 2500 to an equivalent of 50 bases single-end reads, at a depth of approximately 16 million reads per library.
10
Immunoprecipitation of V5- and FLAG-tagged Proteins
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HEK293T cells were used for Co-IP as previously described by our group [2 (link)], but using 1% (v/v) NP-40 lysis buffer. For V5-tagged proteins, anti-V5 agarose beads (Sigma, St Louis, MO, USA) were used. For FLAG tagged proteins, cell lysates were precipitated with anti-FLAG M2 affinity gel (Sigma). Laemmli buffer (Bio-Rad) supplemented with beta-mercaptoethanol was used to elute proteins. Proteins were analyzed by Western blot.
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