MCF7 cells were transfected for 24 h with empty vector and FLAG-TBX2 pcDNA3.1. Whole-cell lysates were prepared with ELB (0.5 mM DTT, 5 mM EDTA pH 8.0, 50 mM HEPES pH 7.5, 0.1% IGEPAL, 250 mM NaCl). For FLAGTBX2 IP, 50 ml of sheep anti-mouse IgG Dynabeads (Invitrogen) were conjugated with 3 μg of FLAG (M2) mouse monoclonal antibody (Sigma) and 3 μg of negative control mouse IgG1 (Dako), and washed and precleared with sheep anti-mouse IgG Dynabeads (Invitrogen) before adding to the antibody-conjugated beads and rotated for 4 h (4 °C). Beads were washed three times with ELB, resuspended in 20 ml of 10× protein sample buffer and boiled (95 °C for 10 min). Protein samples were then analysed by western blot analysis.
Dynabeads sheep anti mouse igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Dynabeads Sheep anti-Mouse IgG are uniform, superparamagnetic beads coated with polyclonal sheep anti-mouse IgG antibodies. These beads are designed for the isolation, purification, or detection of mouse immunoglobulin G (IgG) from various sample types.
Automatically generated - may contain errors
Lab products found in correlation
Glass bead, by Merck Group (2 mentions)
Anti polr2c 1y26, by Abcam (2 mentions)
Cd19 microbeads kit, by Miltenyi Biotec (1 mentions)
Ficoll hypaque, by GE Healthcare (1 mentions)
Ab951, by Abcam (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Anti flag antibody f1804, by Merck Group (1 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
Genechip s cerevisiae tiling 1.0r array, by Thermo Fisher Scientific (1 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (1 mentions)
6870 freezer mill, by SPEX (1 mentions)
Alprenolol, by Merck Group (1 mentions)
Atenolol, by Merck Group (1 mentions)
Isoproterenol, by Merck Group (1 mentions)
Lipopolysaccharide lps from e coli 0111 b4, by Merck Group (1 mentions)
Ici 118 551, by Merck Group (1 mentions)
3h cgp12177, by PerkinElmer (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Flag m2 mouse monoclonal antibody, by Merck Group (1 mentions)
19 protocols using dynabeads sheep anti mouse igg
1
FLAG-TBX2 Immunoprecipitation in MCF7 Cells
2
Isolation of Highly Pure B Lymphocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood samples of healthy individuals were obtained through the blood bank of the University of Torino. PBMCs were obtained by Ficoll‐Hypaque (GE Healthcare Life Sciences, Milan, Italy) centrifugation. B lymphocytes were purified through a first step of negative selection, using anti-CD3, anti-CD16, and anti-CD14 monoclonal antibodies (produced locally) and immunomagnetic bead separation (Dynabeads Sheep anti-Mouse IgG, Life Technologies) followed by positive purification using the CD19 microbeads kit [Miltenyi Biotec S.r.l., Calderara di Reno (BO), Italy]. Flow cytometric analysis showed that these cells were more than 95% CD19+.
3
Isolation and Characterization of STBEV
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dynabeads Sheep Anti-Mouse IgG (50 µL; Life Technologies, CA) were incubated overnight at 4°C with either anti-NEP antibody (6 µg/mL, MA1-19086; Thermo Fisher Scientific), anti-PLAP antibody (6 µg/mL; NDOG2, in-house antibody), or anti-IgG1 isotype control antibody (6 µg/mL, 400153; BioLegend). STBEV from NP placentas were incubated for 10 minutes at 4°C with 10× FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany). Antibodycoated Dynabeads were then incubated overnight at 4°C with 25 µg of blocked STBMV or STBEX in PBS. Unbound sample was washed using PBS and STBEV separated from the Dynabeads by reducing agent, 1× Laemmli Sample Buffer (Bio-Rad Laboratories, CA) and centrifugation at 13 000 RPM. Western blotting was performed on isolated STBEV, and blots were probed using anti-NEP antibody (1:1000, ab951; Abcam) and anti-PLAP antibody (1:1000, NDOG2, in-house antibody).
4
Chromatin Immunoprecipitation and BrdU-IP Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chromatin immunoprecipitation was carried out as previously described [26] (link), [55] (link) with the modification that cells were lysed using a 6870 Freezer/Mill (SPEX, CertiPrep). Briefly, cells were crosslinked by 1% formaldehyde and then washed three times in ice-cold 1× TBS, before being lysed in the Freezer/Mill. Cell lysate was thawed on ice and suspended in lysis buffer. Chromatin was then sheared to a size 300–500 bp by sonication and IP reactions, with anti-FLAG antibody (F1804, Sigma) conjugated to Dynabeads Protein A (Invitrogen), were allowed to proceed over night. After washing and eluting the ChIP fraction from beads, crosslinks were reversed for input and ChIP fractions and DNA was purified. The DNA samples were then processed for sequencing (see below), qPCR or hybridization to microarrays. qPCR was performed using SYBR green (Applied Biosystems) and primers listed in Table S2 on Applied Biosystem 7000 Real-Time PCR System according to the manufacturer's instructions. For ChIP-on-chip, hybridization of ChIP and input fractions to GeneChip S. cerevisiae Tiling 1.0R Array (Affymetrix) was performed as described [26] (link), [55] (link). BrdU-IP was performed as previously described [55] (link) using monoclonal anti-BrdU antibody (clone Bu 20a, Dako) and Dynabeads Sheep Anti-Mouse IgG (Invitrogen).
5
Radioligand Binding Assay for Beta-Adrenergic Receptors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
[3H]-CGP12177 (specific activity 30-60 Ci/mmol, PerkinElmer, USA). Isoproterenol (ISO), atenolol, alprenolol, ICI 118,551 and lipopolysaccharide (LPS, from E. coli0111:B4) (Sigma, St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM) and trypsin (HyClone, Thermo Scientific, Beijing, China), and fetal bovine serum (FBS, Gibco, Invitrogen, Carlsbad, USA). Dynabeads® Sheep anti-Mouse IgG (Invitrogen, Carlsbad, USA). Antibodies against Rab5a (Abcam Hong Kong Ltd, China). Antibodies against phospho-ERK1/2, total ERK1/2 and CD31 (Santa Cruz Biotechnology, Inc., CA). Biotin conjugated BSA (biotin-BSA, Thermo Scientific Pierce Biotechnology, IL, USA).
6
Immunoprecipitation of GFP-tagged Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunoprecipitation, Dynabeads Sheep anti-Mouse IgG (Invitrogen 11031) magnetic beads (30 μL) were incubated at room temperature for 2 hours with 1 μg mouse monoclonal anti-GFP (Invitrogen 3E6 A-11120 ), washed thrice with Wash Buffer (150 mM NaCl, 10 mM HEPES, 1 mM EGTA, and 0.1 mM MgCl2, pH 7.4 with 0.1% Triton X-100) and then incubated with 350 μg protein in 1:1 8M urea with Complete protease inhibitor (Roche 11245200) sample lysis:Buffer A/0.5% Triton X-100 (150 mM NaCl, 10 mM HEPES, 1 mM EGTA, and 0.1 mM MgCl2, with 0.5% Triton X-100 and Complete protease inhibitor (Roche 11245200)). After sample had incubated with beads for 4 hours at 4°C, the beads were washed 6 times with Wash Buffer, and proteins eluted from beads with Laemmli buffer. Samples were then resolved by SDS-PAGE followed by western blot as described above.
7
CD155-Mediated Modulation of CLL Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Modulation of the signaling triggered by CD155 binding either TIGIT or CD226 was performed both in the short term, to evaluate its inference on αIgM-mediated pBTK induction, and in the long term, to investigate the impact on CpG/IL-15-induced CLL proliferation. For the short-term experiments, cells were pre-treated in ice for 1 h with 5 µg/mL recombinant human (rh)TIGIT-Fc or with αTIGIT or αCD226 blocking monoclonal antibodies (5 µg/106 cells) for 30 min and then with rhCD155-Fc (5 µg/mL) for 1 h, before αIgM stimulation. For the long-term experiments, to prevent internalization, αTIGIT and αCD226 blocking monoclonal antibodies were coated onto magnetic beads and rhTIGIT-Fc and rhCD155-Fc chimeras were immobilized onto a cell culture plate. Briefly, 10x106 Dynabeads Sheep anti-Mouse IgG (Invitrogen, Thermofisher) were washed twice with phosphate-buffered saline, 0.1% bovine serum albumin and then coated with 1.5 μg of either antibody, by incubating overnight at 4°C on a rotating wheel, following the manufacturer’s instructions. Coated beads were used to treat CLL cells by pre-mixing them at a 2:1 bead:cell ratio immediately before plating the cells in a 96-well plate. In parallel, 96-well plates were coated overnight at 4°C with 1 μg/well of rhTIGIT-Fc or rhCD155-Fc chimeras.
8
Purification of CXCR4-positive Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To purify CXCR4-positive cells, Dynabeads™ sheep anti-mouse IgG (Invitrogen, Carlsbad, USA), a magnetic holder, and purified mouse anti-human CD184 (clone 12G5, cat: 555972, BD Pharmingen™, AB_396265) were used. Approximately 10x106 cells were centrifuged (5 min, 400 g), and washed once in DPBS supplemented with 2% FBS. Cells were then incubated for 30 minutes in 1 mL DPBS + 2% FBS, to which 25 μL of the purified antibody was added. Cells were washed twice in DPBS + 2% FBS (5 min, 400 g), and resuspended in 6 mL DPBS + 2% FBS. In the meantime, 10x106 beads were washed twice in DPBS + 2%FBS according to the manufacturer’s instructions, and finally resuspended in 3 mL DPBS + 2% FBS. The cell and bead suspensions were gently mixed, and the mixture was incubated for 30 min with constant mixing on a rotator. The mixture was washed twice witch DPBS + 2% FBS (magnetic holder) and the beads with cells were resuspended in 10 mL selective McCoy’s 5a medium supplemented with 10% fetal bovine serum (FBS), 1 mM streptomycin, and 1 mM penicillin.
9
Whole-Cell Protein Extraction and Immunoprecipitation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-cell protein extracts and protein immunoprecipitation were performed as described [62 (link)]. Briefly for protein extract preparation, 150 mL of cells growing exponentially (OD600 0.6–0.8) were centrifuged, pelleted, and resuspended in 0.3 mL of lysis buffer (50 mM HEPES [pH 7.5], 120 mM NaCl, 1 mM EDTA, 0.3% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS)) supplemented with 1X protease inhibitor cocktail (Complete; Roche; Basel, Switzerland), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM sodium orthovanadate, and 1 mM sodium fluoride. Cell disruption was carried out by vortexing (3 cycles, 5 min each) at 4 °C using 0.2 mL of glass beads (425–600 µm; Sigma, Darmstadt, Germany). For the Rpb3 immunopurification, 1 µg of anti-Rpb3 antibody (anti-POLR2C;1Y26, Abcam, Cambridge, UK) was coupled to 40 µL of Dynabeads Sheep-anti-Mouse IgG (Invitrogen, Waltham, MA, USA) per sample, and 2 mg of a whole-cell protein extract were used for each immunoprecipitation. The affinity-purified proteins were released from the beads by boiling for 10 min and were analyzed by Western blotting with different antibodies.
10
Protein Extraction and Immunoprecipitation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein whole cell extracts and protein immunoprecipitation were performed as described [63] . Briefly for protein extract preparation, 150 ml of cells growing exponentially (OD 600 0.6-0.8) were centrifuged, pelleted and resuspended in 0.3 ml of lysis buffer (50 mM HEPES [pH 7.5], 120 mM NaCl, 1 mM EDTA, 0.3% 3-[(3cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS)) supplemented with 1X protease inhibitor cocktail (Complete; Roche), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM sodium orthovanadate, and 1 mM sodium fluoride. Cell disruption was carried out by vortexing (3 cycles, 5 min each) at 4ºC using 0.2 ml of glass beads (425-600 µm; Sigma). For the Rpb3 immunopurification, 1µg of anti-Rpb3 antibody (anti-POLR2C;1Y26, Abcam) was coupled to 50 µl of Dynabeads Sheep-anti-Mouse IgG (Invitrogen) per sample, and 2 mg of a whole cell protein extract were used for each immunoprecipitation. A similar procedure was followed for TAP pull down, with 50 µl of Dynabeads-Pan Mouse (Invitrogen). The affinity-purified proteins were released from the beads by boiling for 10 min and were analysed by western blotting with different antibodies.
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!