To test the effect of RANTES (regulated on activation, normal T-cell expressed and secreted) on p24 release from HIV-1 R5SF162 infected PBMCs, human recombinant RANTES was added to diluted (1%) SP from uninfected donors to reach a final concentration of 5 or 10 pg/ml (corresponding to 500 and 1000 pg/ml, respectively, in undiluted SP). Conversely, SPs from infected donors were depleted of RANTES: after a preclearing of IgGs by incubation with an excess (1 mg/ml) of GammaBind Sepharose beads (GE Healthcare, Little Chalfont, Buckinghamshire, UK) for 1 h at 4°C with rotation, the beads were removed by centrifugation for 3 min at 1500 rpm, and anti-RANTES mAbs (10 mg/ml) were added for 1 h on ice. A second incubation with GammaBind Sepharose beads was performed, and immunocomplexes bound to beads removed by centrifugation. Treated samples were tested by ELISA (R&D Systems) to confirm RANTES depletion.
Gammabind sepharose beads
Manufactured by GE Healthcare
Sourced in United Kingdom, United States
GammaBind Sepharose beads are a ready-to-use affinity chromatography medium designed for the purification of immunoglobulins. The beads consist of cross-linked agarose that is covalently coupled with recombinant protein G.
Automatically generated - may contain errors
Lab products found in correlation
Glutathione sepharose 4b beads, by GE Healthcare (2 mentions)
Millipore immobilon ecl reagents, by Merck Group (2 mentions)
Anti epha2 antibody, by Thermo Fisher Scientific (2 mentions)
Anti phosphotyrosine antibody, by Merck Group (2 mentions)
Gapdh antibody, by Abcam (1 mentions)
Sepharose beads, by GE Healthcare (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
6 protocols using gammabind sepharose beads
1
RANTES Effect on HIV-1 Infection in PBMCs
2
GST-CAPK and HA-KIF3A Pulldown Protocol
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Forty eight hours after transfection, cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 2 mM EGTA, complete protease inhibitors [Roche], 10 mM sodium orthovanadate, 5 mM sodium fluoride, 10 mM sodium pyrophosphate, 10 mM β-glycerophosphate, and 1 µM microcystin LR). Cell lysate was cleared by centrifugation. GST-CAPK proteins were pulled down from cell lysate using glutathione Sepharose 4B beads (GE Healthcare, Chicago, IL, USA) following the manufacturer’s instruction. HA-KIF3A proteins were immunoprecipitated from cell lysate using the HA antibody, and captured on GammaBind Sepharose beads (GE Healthcare).
Cell extracts or Sepharose beads were boiled for 5 min in an equal volume of 2X Laemmli sample buffer (120 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% β-mercaptoethanol, 0.02% bromophenol blue) and loaded on an SDS gel. Samples were transferred to a PVDF (polyvinylidene difluoride) membrane and blocked for one hour in 5% dry milk before primary antibody incubation in TBS containing 0.1% Tween-20 and 5% bovine serum albumin (BSA) for 90 min at room temperature or overnight at 4 °C. This was followed by extensive rinses and one hour incubation with horseradish peroxidase (HRP)-conjugated secondary antibody. Chemiluminescence signals were developed using Millipore Immobilon ECL reagents (EMD Millipore, Burlington, MA, USA).
Cell extracts or Sepharose beads were boiled for 5 min in an equal volume of 2X Laemmli sample buffer (120 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% β-mercaptoethanol, 0.02% bromophenol blue) and loaded on an SDS gel. Samples were transferred to a PVDF (polyvinylidene difluoride) membrane and blocked for one hour in 5% dry milk before primary antibody incubation in TBS containing 0.1% Tween-20 and 5% bovine serum albumin (BSA) for 90 min at room temperature or overnight at 4 °C. This was followed by extensive rinses and one hour incubation with horseradish peroxidase (HRP)-conjugated secondary antibody. Chemiluminescence signals were developed using Millipore Immobilon ECL reagents (EMD Millipore, Burlington, MA, USA).
3
EphA2 Receptor Tyrosine Kinase Activation
4
Ephrin-A1 Fc Stimulation and EphA2 Phosphorylation
5
Immunoprecipitation and Western Blot Analysis
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Forty-eight hours after transfection, cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 2 mM EGTA, complete protease inhibitors (Roche), 10 mM sodium orthovanadate, 5 mM sodium fluoride, 10 mM sodium pyrophosphate, 10 mM β–glycerophosphate, and 1 µM microcystin LR). Cell lysate was cleared by centrifugation. GST-CILK1 proteins were pulled down from cell lysate using Glutathione Sepharose 4B beads (GE Healthcare) following the manufacturer’s instructions. HA-KIF3A proteins were immunoprecipitated from cell lysate using HA antibody and captured on GammaBind Sepharose beads (GE Healthcare).
Cell extracts or Sepharose beads were boiled for 5 min in an equal volume of 2X Laemmli sample buffer (120 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% β-mercaptoethanol, 0.02% bromophenol blue) and loaded on an SDS-gel. Samples were transferred to a PVDF membrane and blocked for one hour in 5% dry milk before primary antibody incubation in TBS containing 0.1% Tween-20 and 5% bovine serum albumin (BSA) for 90 min at RT or overnight at 4°C. This was followed by extensive rinses and one-hour incubation with horseradish peroxidase (HRP)-conjugated secondary antibody. Chemiluminescence signals were developed using Millipore Immobilon ECL reagents from EMD Millipore (Burlington, MA, USA).
Cell extracts or Sepharose beads were boiled for 5 min in an equal volume of 2X Laemmli sample buffer (120 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% β-mercaptoethanol, 0.02% bromophenol blue) and loaded on an SDS-gel. Samples were transferred to a PVDF membrane and blocked for one hour in 5% dry milk before primary antibody incubation in TBS containing 0.1% Tween-20 and 5% bovine serum albumin (BSA) for 90 min at RT or overnight at 4°C. This was followed by extensive rinses and one-hour incubation with horseradish peroxidase (HRP)-conjugated secondary antibody. Chemiluminescence signals were developed using Millipore Immobilon ECL reagents from EMD Millipore (Burlington, MA, USA).
6
Immunoprecipitation and Western Blot of KIF3A
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Forty eight hours after co-transfection of GST-CILK1 and FLAG-KIF3A, HEK293T cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 2 mM EGTA, complete protease inhibitors (Roche, Basel, Switzerland), 10 mM sodium orthovanadate, 5 mM sodium fluoride, 10 mM sodium pyrophosphate, 10 mM β–glycerophosphate, and 1 µM microcystin LR). Cell lysate was cleared by centrifugation. FLAG-KIF3A proteins were immunoprecipitated from cell lysate using FLAG-tag rabbit monoclonal antibody and captured on GammaBind Sepharose beads (GE Healthcare, Chicago, IL, USA).
Sepharose beads were boiled for 5 min in an equal volume of 2× Laemmli sample buffer (120 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% β-mercaptoethanol, 0.02% bromophenol blue) and loaded on an SDS gel. Samples were transferred to a polyvinylidene fluoride (PVDF) membrane and blocked for one hour in 5% dry milk before primary antibody incubation in Tris-buffered saline (TBS) containing 0.1% Tween-20 and 5% bovine serum albumin (BSA) overnight at 4 °C. This was followed by extensive rinses and one-hour incubation with horseradish peroxidase (HRP)-conjugated secondary antibody. Chemiluminescence signals were developed using Millipore Immobilon enhanced chemiluminescence (ECL) reagents (EMD Millipore, Burlington, MA, USA).
Sepharose beads were boiled for 5 min in an equal volume of 2× Laemmli sample buffer (120 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% β-mercaptoethanol, 0.02% bromophenol blue) and loaded on an SDS gel. Samples were transferred to a polyvinylidene fluoride (PVDF) membrane and blocked for one hour in 5% dry milk before primary antibody incubation in Tris-buffered saline (TBS) containing 0.1% Tween-20 and 5% bovine serum albumin (BSA) overnight at 4 °C. This was followed by extensive rinses and one-hour incubation with horseradish peroxidase (HRP)-conjugated secondary antibody. Chemiluminescence signals were developed using Millipore Immobilon enhanced chemiluminescence (ECL) reagents (EMD Millipore, Burlington, MA, USA).
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