Samples from fractions 3 to 5 (TGN/Golgi-containing fractions) were pooled, incubated with 1% Triton X-100 on ice for 10 min, and centrifuged at 16,100g for 10 min. The resulting supernatants were collected, and ~150 ng of WT HPV16.L2F PsV was added per 1 ml of the uninfected sample lysate (called control). The samples were then incubated with anti-FLAG M2 antibody (~8 μg of antibody per 1 ml of lysate) (F3165; Millipore Sigma) at 4°C overnight, and the immune complex was captured with protein G–coated magnetic beads (Invitrogen, 10003D) at 4°C for 1 hour. Beads were washed four times with 0.1% Triton X-100 in HBS buffer. Bound proteins were eluted with 1% SDS in HN buffer (50 mM Hepes and 150 mM NaCl) and denatured by incubating at 90°C for 10 min. A portion of eluate was analyzed by SDS-PAGE followed by immunoblotting to confirm that equivalent amounts of L2-3xFLAG were precipitated from WT or R302/5A-infected conditions or control. The remaining eluate was treated with 10% trichloroacetic acid and incubated on ice for 10 min. The sample was subjected to centrifugation, and the precipitated material washed twice with acetone. The precipitate was subject to mass spectrometry analysis at the Taplin Mass-Spectrometry Core Facility (Harvard Medical School). Liquid chromatography–tandem mass spectrometry was performed using an Orbitrap mass spectrometer (Thermo Fisher Scientific).
Protein g coated magnetic beads
Manufactured by Thermo Fisher Scientific
Protein G-coated magnetic beads are a type of laboratory equipment used in various biomolecular applications. These beads are coated with the protein G, which has a high affinity for the Fc region of immunoglobulin (Ig) molecules. The magnetic properties of the beads allow for easy separation and manipulation of the bound Ig molecules from complex samples, such as cell lysates or serum.
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Lab products found in correlation
Mouse pcsk9 elisa kit, by R&D Systems (2 mentions)
Clarity or clarity max substrate, by Bio-Rad (2 mentions)
Rat anti ha antibody, by Roche (2 mentions)
3xflag peptide, by Merck Group (2 mentions)
Caveolin 2, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Cell Signaling Technology (1 mentions)
Caveolin 1, by Cell Signaling Technology (1 mentions)
Kingfisher 96, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 rslcnano, by Thermo Fisher Scientific (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Sodium dodecyl sulfate (sds), by Thermo Fisher Scientific (1 mentions)
Np 40, by Thermo Fisher Scientific (1 mentions)
Disodium hydrogen phosphate dihydrate, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
15 protocols using protein g coated magnetic beads
1
Proteomic Profiling of HPV16 L2 Interactome
2
Chromatin Immunoprecipitation Protocol
3
Magnetic Sorting of Plasmodium falciparum
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The Plasmodium falciparum clone 3D7 and nine laboratory-adapted parasite isolates from severe and non-severe malaria patients in Kilifi, Kenya (sampled between 2009 and 2010), were cultured in vitro according to standard procedures20 (link) and cryopreserved at the late trophozoite stage for use in subsequent assays. To select for MGD21-reactive infected erythrocytes (IE), cultured IE were incubated with MGD21 for 20 min at room temperature, washed, and rotated with Protein G-coated magnetic beads (Life Technologies) for 30 min at room temperature. Following magnetic sorting, enriched (MGD21+) and depleted (MGD21-) fractions were returned to in vitro culture.
4
Magnetic Sorting of Plasmodium falciparum
5
Quantifying Plasma PCSK9 Levels
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Plasma PCSK9 level was tested by a mouse PCSK9 ELISA kit (R&D Systems) by comparing experimental sera samples to an internal standard curve according to manufacturer’s protocol. PCSK9 level was quantified both before and after removal of immunoglobulin using Protein G-coated magnetic beads (Life Technologies). Plasma samples were incubated for 10 min with magnetic Protein G beads at room temperature. The Protein G beads were isolated using a magnet, and the immunoglobin-free supernatant was then used directly in PCSK9 ELISA kit.
6
PCSK9 Quantification in Plasma
7
Phosphoprotein Analysis by Immunoprecipitation
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For immunoprecipitation experiments, cells were lysed in CPBS buffer (6 mM CHAPS in PBS [pH 7.4]). Lysates were incubated with the indicated antibody (1:50, 14 hr, 4°C), and the protein-antibody complex was precipitated by centrifugation after incubation with protein G-coated magnetic beads (Dynal, 2 hr, 4°C). The immunoprecipitated material was washed twice in CPBS and resuspended in SDS/PAGE loading buffer (NuPAGE), boiled, and loaded on 4%–12% gels (NuPAGE). For phosphorylation studies, total cell lysates were loaded on a phosphoprotein binding column (QIAGEN) as previously described (Cereghetti et al., 2008 (link)). Flow-through (unphosphorylated) and eluted (phosphorylated) proteins were collected and concentrated and 20 μg of proteins were separated by 4%–12% SDS-PAGE.
8
Immunoprecipitation and Western Blot Analysis
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HEK-293T cells were harvested 24h post-transfection. For the production of whole-cell lysates, cells were lysed in 300 μl of lysis buffer (50 mM Tris at pH 7.4, 1% NP-40 [v/v], 150 mM NaCl , 1mM GTP, 10mM MgCl2, a protease inhibitor cocktail [1x EDTA-free protease inhibitors, Sigma], 10mM NaF, 1mM Na3VO4, 2μM Bortezomib) at 4°C. 60 μl of lysates was saved as input and the rest was subject to immunoprecipitation.
For immunoprecipitation, rat anti-HA antibodies (Roche) were coupled covalently to protein G-coated magnetic beads (Dynal) using dimethyl pimelimidate. Anti-HA-coupled beads were added to the clarified lysate. After 1.5h binding at 4°C, the beads were washed 3x 5min at 4°C with lysis buffer and eluted with 2x SDS sample buffer at 65°C for 30 min.
Western blot detection was performed by enhanced chemiluminescence using HRPcoupled secondary antibodies and the Clarity or Clarity Max substrates (Bio-Rad). The signal was digitized using Amersham 600RGB imager as 16-bit grayscale TIFF files. Quantitative analysis of band intensities was performed with ImageJ.
For immunoprecipitation, rat anti-HA antibodies (Roche) were coupled covalently to protein G-coated magnetic beads (Dynal) using dimethyl pimelimidate. Anti-HA-coupled beads were added to the clarified lysate. After 1.5h binding at 4°C, the beads were washed 3x 5min at 4°C with lysis buffer and eluted with 2x SDS sample buffer at 65°C for 30 min.
Western blot detection was performed by enhanced chemiluminescence using HRPcoupled secondary antibodies and the Clarity or Clarity Max substrates (Bio-Rad). The signal was digitized using Amersham 600RGB imager as 16-bit grayscale TIFF files. Quantitative analysis of band intensities was performed with ImageJ.
9
Immunoprecipitation and Western Blot Analysis
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HEK-293T cells were harvested 24h post-transfection. For the production of whole-cell lysates, cells were lysed in 300 μl of lysis buffer (50 mM Tris at pH 7.4, 1% NP-40 [v/v], 150 mM NaCl , 1mM GTP, 10mM MgCl2, a protease inhibitor cocktail [1x EDTA-free protease inhibitors, Sigma], 10mM NaF, 1mM Na3VO4, 2μM Bortezomib) at 4°C. 60 μl of lysates was saved as input and the rest was subject to immunoprecipitation.
For immunoprecipitation, rat anti-HA antibodies (Roche) were coupled covalently to protein G-coated magnetic beads (Dynal) using dimethyl pimelimidate. Anti-HA-coupled beads were added to the clarified lysate. After 1.5h binding at 4°C, the beads were washed 3x 5min at 4°C with lysis buffer and eluted with 2x SDS sample buffer at 65°C for 30 min.
Western blot detection was performed by enhanced chemiluminescence using HRPcoupled secondary antibodies and the Clarity or Clarity Max substrates (Bio-Rad). The signal was digitized using Amersham 600RGB imager as 16-bit grayscale TIFF files. Quantitative analysis of band intensities was performed with ImageJ.
For immunoprecipitation, rat anti-HA antibodies (Roche) were coupled covalently to protein G-coated magnetic beads (Dynal) using dimethyl pimelimidate. Anti-HA-coupled beads were added to the clarified lysate. After 1.5h binding at 4°C, the beads were washed 3x 5min at 4°C with lysis buffer and eluted with 2x SDS sample buffer at 65°C for 30 min.
Western blot detection was performed by enhanced chemiluminescence using HRPcoupled secondary antibodies and the Clarity or Clarity Max substrates (Bio-Rad). The signal was digitized using Amersham 600RGB imager as 16-bit grayscale TIFF files. Quantitative analysis of band intensities was performed with ImageJ.
10
FGFR1-Mediated Rickettsial Infection Dynamics
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ECs were infected with SFG rickettsiae for 1 hour and whole cell lysates were prepared using modified radio immunoprecipitation assay (RIPA) buffer supplemented with a protease inhibitor cocktail (Cell Signaling). Immunoprecipitation (IP) was carried out using an FGFR1 antibody (Abgent) covalently cross-linked to protein G-coated magnetic beads (Thermo Fisher) using mouse IgG as a negative control. The beads were then washed thoroughly with RIPA buffer and the samples thus prepared were analyzed by SDS gel electrophoresis and Western Blotting using an OmpA antibody (kindly provided by Dr. Donald H. Bouyer, UTMB) at 1:1,000 dilution. In other experiments, caveolin-1 or caveolin-2 (Cell Signaling), phospho-FGFR1 (Y653/654, Cell Signaling), and α-Tubulin (Accurate Chemical and Scientific) antibodies were used in conjunction with appropriate HRP-conjugated secondary antibodies.
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