Catalog numbers are listed in parentheses. The following reagents were purchased from Sigma-Aldrich: β-mercaptoethanol (M6250), dithiothreitol (D0632), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; M2128), dimethyl sulfoxide (DMSO; M81802), doxorubicin (44583), Gly-Pro (G3002), MG132 (M7479), N-acetyl-cysteine (A7250), phenylmethanesulfonyl fluoride (PMSF; 329-98-6), phosphatase inhibitor cocktail 2 (P5726), proline (P0380), propidium iodide (PI; P-4170), puromycin (P8833), rhodamine 123 (62669-70-9), Tempol (581500) and trypan blue (T8154). Blasticidin (R21001), CM-H2DCFDA (C6827), MluI restriction enzyme (ER0561), RNase (AM2286) and SgfI restriction enzyme (FD2094) were purchased from Thermo Fisher Scientific. A protease inhibitor cocktail (11-836-153-001) was purchased from Roche Applied Science. G-sepharose beads (17-6002-35) was purchased from GE Healthcare. Hydrogen peroxide 3% USP (F0010) was purchased from Hydrox Laboratories. Matrigel (356237) was purchased from Corning. Phos-tag acrylamide (304-93562) was purchased from Wako. Pifithrin-α (1267) was purchased from R&D Systems. Sodium dodecyl sulfate (SDS; 161-0301) was purchased from Bio-Rad. Ubiquitin aldehyde (UbAl; BML-UW8450) and Z-VAD-FMK (ALX-260-020-M001) were purchased from Enzo Life Sciences.
G sepharose beads
Manufactured by GE Healthcare
Sourced in Germany, Sweden
G-sepharose beads are a solid-support material used in affinity chromatography for the purification of biomolecules. They consist of agarose beads to which Protein G, a bacterial protein with high affinity for immunoglobulins, has been covalently coupled. G-sepharose beads can be used to capture and isolate antibodies and antibody-containing samples from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Blasticidin, by Thermo Fisher Scientific (1 mentions)
Matrigel 356237, by Corning (1 mentions)
Phos tag acrylamide, by Fujifilm (1 mentions)
Sodium dodecyl sulfate (sds), by Bio-Rad (1 mentions)
Ecl plus kit, by GE Healthcare (1 mentions)
Anti src and anti phospho src antibodies, by Cell Signaling Technology (1 mentions)
Anti fak, by BD (1 mentions)
Alexa fluor 488 conjugated goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Anti cortactin, by Merck Group (1 mentions)
Anti phosphotyrosine, by Merck Group (1 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated goat anti mouse, by Promega (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Mntmpyp, by Merck Group (1 mentions)
Protein a g agarose beads, by GE Healthcare (1 mentions)
Anti egfr, by Abcam (1 mentions)
Rt pcr kit, by Takara Bio (1 mentions)
Fibronectin, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
24 protocols using g sepharose beads
1
Comprehensive Cellular Assay Protocol
2
Antibody Characterization for Cell Signaling
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The following mouse monoclonal antibodies were used: anti-FAK (BD Transduction Laboratories, Le Pont de Claix, France), anti-cortactin (Millipore, Molsheim, France), anti-Tks-5 (Tebu-bio, Le Perray-en-Yvelines, France), anti-Tks-5 PX domain, anti-Tks-5 SH3 domain, anti-phosphotyrosine (Millipore), anti-talin, anti-paxillin, anti-phospho-Y397 FAK (Invitrogen, Camarillo, CA, USA) and anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA). Anti-Src and anti-phospho-Src anti-bodies were from Cell Signalling (Danvers, MA, USA) or Invitrogen (Grand Island, NY, USA). Horseradish peroxidase-conjugated goat anti-mouse was from Promega (Madison, WI, USA). Actin antibody conjugated to Alexa fluor 647 was from Santa Cruz (Heidelberg, Germany). PP2 was from Calbiochem (Billerica, MA, USA) and MnTMPyP from MERCK Biosciences (Darmstadt, Germany). G-sepharose beads and ECLplus kit were from GE healthcare (Freiburg, Germany). MnPyP, CM-H2DCF-DA, Alexa Fluor 488-conjugated goat anti-mouse, Alexa-405-labelling kit and Lipofectamine 2000 were from Invitrogen. Gelatin from porcine skin and mouse monoclonal Ab was from Sigma-Aldrich.
3
Nuclear and Cytoplasmic Fractionation Protocol
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Subcellular fractionation was prepared as previously described [28 (link)]. For obtaining the nuclear fraction, the pellets were incubated in the ice-cold nuclei isolation buffer (10 mM HEPES pH 7.9, 1,5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 1 % Triton X-100, 15 % protease inhibitor cocktail Complete Mini, 1 mM PMSF). After the separation steps, the nuclear and cytoplasmic fractions were obtained and subjected to immunoblot analysis. Antibodies against β-tubulin and Lamin B were used to validate the purity of the cytoplasmic and nuclear fractions, respectively. For immunoprecipitation (IP) assay, antibody against PIP5K1α was used to pull down the immunocomplexes, and antibody to IgG (BD Biosciences, San Jose, CA, USA) was used as a negative control. 500 μg of freshly prepared protein lysates were incubated with the antibody of interests and 30 μl of G-sepharose beads (GE Healthcare) for 3 hours at 4°C. The samples were then washed in RIPA buffer and subjected to immunoblot analysis.
4
EGF Stimulation and SOS1 Binding
5
Investigating GRP78 Signaling Pathways
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The following antibodies were used: anti-Src, anti-pSrcY416, anti-FAKpY397, anti-pPaxillin Y118, anti-Paxillin, anti-pCortactin Y486, anti-pCortactin Y466, anti-Cortactin were all from life technologies; Anti-GRP78 N20, antiGRP78 C20 (for antibody blocking), anti-β-actin were from Santa Cruz. Anti-GRP78 (for immunoprecipitation and in cell western analysis), anti-pEGFR Y1101, anti-pEGFR Y1068, anti-pEGFR Y845, anti-EGFR, anti-p-Tyr and rabbit isotype IgG were obtained from Abcam. Anti-EGFP was obtained from Origen. All the secondary antibodies except for IRDYE680RD-conjugated antibody (LI-COR) were all from abcam. α2M was purchased from Sigma-Aldrich and activated as previously reported [18 (link)]. PP2, lipofectamine 2000, TRITC-WGA and fibronectin were from life technologies. Protein A/G agarose beads and G-sepharose beads were purchased from GE healthcare. RT-PCR kit was from Takara. Plasma protein isolation kit was purchased from Pierce. GRP78-EGFP recombinant and corresponding pEGFP-N1 were kindly given by the Cell Biology Department of China Medical University.
6
Cx43 Immunoprecipitation and Proteomic Analysis
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Samples were prepared in RIPA buffer, containing protease inhibitors (protease inhibitor cocktail (Roche) and 2 mM sodium orthovanadate) and supplemented with 0.5% TX100. To IP Cx43, 0.25 µg goat anti-Cx43 (AB0016, SICGEN) were incubated with 500 µg total protein lysates overnight, at 4°C under agitation. 8 µg of dry protein G Sepharose beads (GE Healthcare) were added to the samples for 2 h, at 4°C under agitation. Beads were washed in RIPA buffer, followed by elution in Laemmli buffer and denaturation at 95°C, for 5 min.
For proteomic analysis, 3 mg total protein nuclear lysates were used for IP. Beads were washed three times in 1 ml trypsin digestion buffer (20 mM Tris-HCl pH 8.0, 2 mM CaCl2). MS analysis was performed in VIB Proteomics Core, VIB Center for Medical Biotechnology (Ghent, Belgium).
For proteomic analysis, 3 mg total protein nuclear lysates were used for IP. Beads were washed three times in 1 ml trypsin digestion buffer (20 mM Tris-HCl pH 8.0, 2 mM CaCl2). MS analysis was performed in VIB Proteomics Core, VIB Center for Medical Biotechnology (Ghent, Belgium).
7
ChIP-Seq Protocol for H3K27ac Analysis
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We performed chromatin immunoprecipitation (ChIP) as described in Ref.38 (link). A2058 cells were cross-linked for 10 min by 1% formaldehyde and fragmented by a sonicator (UD-211; TOMY SEIKO, Tokyo, Japan). The samples were immunoprecipitated with 4 μg of antibodies against H3K27ac (05-1334, Merck Millipore, Billerica, MA, USA). We used protein A-sepharose beads and G-sepharose beads (GE Healthcare, Barrington, Ill, USA) to immunoprecipitate the fragments. Chromatin immunoprecipitation sequence (ChIP-Seq) libraries were made using a KAPA Hyper Prep kit (Roche, Wilmington, MA, US) with 1 ng of ChIP DNA. Libraries were size-selected prior to PCR amplification using AMPure XP beads (Beckman Coulter, Brea, CA, USA). Multiplexed libraries were run on an Illumina HiSeq 2500 genome sequencer (Illumina Inc. San Diego, CA, USA) using the 50-base pair single read method. The nucleotide sequence data reported are available in the DDBJ Sequenced Read Archive under the accession numbers DRX230605 and DRX230606.
8
Immunoprecipitation and Western Blot Analysis
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Whole cell lysate (100 μg of total protein) was added to 1/200 volume of each rabbit antibody and adjusted to 680 μL of volume with the lysis buffer. The antibodies used are listed in the Online Supplementary Methods. Rabbit serum-saturated protein G-Sepharose beads (20 μL) (GE Health Care, Uppsala, Sweden) were added to each lysate, and immunoprecipitation was performed for 1 h while rotating at 4°C. After incubation, the beads were washed three times with ice-cold lysis buffer and boiled for 3 min with 20 μL/tube of loading buffer. The supernatant was subjected to western blot analysis.
9
Kinase Assay with ABL1 Immunoprecipitation
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For the kinase assay, ABL1 proteins were immunoprecipitated at a ratio of 3.5 μL ABL1 antibody per 0.8 mg whole cell extract and 65 μL slurry of G-sepharose beads (GE-Healthcare). For each kinase reaction with 5 mg FSBA-treated and desalted cellular protein fraction, an equivalent of ABL1-Kin− and ABL1-PP immunopurified from 3.2 mg total HEK293 cell lysate was prepared. Beads were washed 2× with IP-buffer and 1× with KA buffer (40 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT), pooled and resuspended in KA buffer. The beads were equally split into eppendorf tubes, 5 mg FSBA-treated cellular protein fraction added and the kinase reaction initiated by adding ‘light’ (SIGMA-Aldrich) or ‘heavy’ 18O2-ATP (Organisch-Chemisches Institut, UZH, Switzerland)29 (link) at a final concentration of 1 mM. After incubation for 30, 90 and 150 min at RT (~23 °C) on a rotary wheel, the reaction was quenched by the addition of solid urea to a final concentration of 8 M. Samples were vortexed and stored on ice.
10
Immunoprecipitation of Amyloid Oligomers
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For immunoprecipitation (IP), 400 µg of total protein from brain extract (10% w/v) was diluted in a total volume of 1000 µL. G sepharose beads (GE Healthcare) were washed 3× with PBS and 50 µL per 500‐µL diluted brain homogenate were incubated with 1 µL of Aβ antibody 3552 (kindly provided by E. Kremmer, Ludwig Maximilians University, Munich, Germany) 33 or amyloid oligomeric‐specific antibody A11 (A11, Millipore) overnight by 4°C shaking head over heel. The supernatant was reused. This experiment was repeated until the remaining oligomers resulted in greatly reduced band intensities as detected by western blot probed with Aβ antibody 6E10 (Covance). The cleared supernatant was used for the experiments.
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