Epidermal lysates were prepared as for Western blot analysis. 100 µg of epidermal lysate was incubated with 10 µg of the indicated antibody. The total volume of the lysate/antibody mixture was adjusted to 1,000 µL with lysis buffer to allow for appropriate mixing and rotated at 4°C overnight. Lysate/antibody mixture was then mixed with 50 µL of protein agarose A/G (sc-2003 Santa Cruz Biotechnology, Santa Cruz, CA) for 6 h. Lysate/antibody/protein A/G agarose mixture was then centrifuged at 8,000g for 10 min to sediment the protein A/G agarose. The pellet was washed with 0.1% tween in PBS and then sedimented at 8,000g for 10 min three times to wash any non-specific binding from the pellet. After three washes the immunoprecipitate was boiled for 5 min in 20 µL Protein Loading Buffer Blue (Cat # EC-886, National Diagnostics, Atlanta, GA). Immunoprecipitates were then treated as described above under Western Blot analysis method.
Protein a g agarose
Manufactured by Santa Cruz Biotechnology
Sourced in United States, China
Protein A/G agarose is a laboratory reagent used for the purification and isolation of immunoglobulins and antibodies from biological samples. It consists of agarose beads covalently coupled with Protein A and Protein G, which are bacterial surface proteins that bind to the Fc region of antibodies. This affinity-based resin can be used in various chromatographic techniques to capture and enrich antibodies from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (17 mentions)
Protease inhibitor cocktail, by Roche (16 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (9 mentions)
Cell lysis buffer, by Cell Signaling Technology (7 mentions)
Laemmli sample buffer, by Bio-Rad (6 mentions)
Protease inhibitor, by Roche (6 mentions)
Gapdh, by Santa Cruz Biotechnology (5 mentions)
2 mercaptoethanol, by Merck Group (5 mentions)
Anti flag, by Merck Group (5 mentions)
Sample buffer, by Merck Group (5 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Protein loading buffer blue, by National Diagnostics (4 mentions)
Anti ha, by Roche (4 mentions)
Anti v5, by Thermo Fisher Scientific (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Complete protease inhibitor cocktail, by Roche (3 mentions)
Protease inhibitors cocktail, by Merck Group (3 mentions)
Ezview red anti flag m2 affinity gel, by Merck Group (3 mentions)
321 protocols using protein a g agarose
1
Immunoprecipitation of Epidermal Proteins
2
Immunoprecipitation of Epidermal Proteins
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Epidermal lysates were prepared as for Western blot analysis. 100 µg of epidermal lysate was incubated with 10 µg of the indicated antibody. The total volume of the lysate/antibody mixture was adjusted to 1,000 µL with lysis buffer to allow for appropriate mixing and rotated at 4°C overnight. Lysate/antibody mixture was then mixed with 50 µL of protein agarose A/G (sc-2003 Santa Cruz Biotechnology, Santa Cruz, CA) for 6 h. Lysate/antibody/protein A/G agarose mixture was then centrifuged at 8,000g for 10 min to sediment the protein A/G agarose. The pellet was washed with 0.1% tween in PBS and then sedimented at 8,000g for 10 min three times to wash any non-specific binding from the pellet. After three washes the immunoprecipitate was boiled for 5 min in 20 µL Protein Loading Buffer Blue (Cat # EC-886, National Diagnostics, Atlanta, GA). Immunoprecipitates were then treated as described above under Western Blot analysis method.
3
Protein Expression Analysis via IP-Western Blot
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For immunoprecipitation (IP), the cell lysates, prepared with agarose protein A+G (Santa cruz biotechnology, Dallas, Texas, USA) to eliminate nonspecific binding, were incubated with primary antibody overnight at 4°C, and mixed with agarose protein A+G for 6 h at 4°C. After washed three times by lysis buffer, the loading buffer was added and the agarose protein A+G were removed by centrifugation. Western blot was performed to analyze protein expression. For western blot, it was performed according to the standard method, briefly, 30 ug total protein was loaded on gel, after the progress of running the gel, transferring the protein, and incubating with antibodies, the protein image was acquired by the chemiluminescence detection system.
4
Immunoprecipitation and Immunoblotting of Ubiquitin
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Immunoprecipitation was performed according to a previously described method [42 (link), 43 (link)]. Lysates were pre-cleaned in lysis buffer with protein A/G agarose (Santa Cruz Biotechnology) for 1 h at 4°C and subsequently centrifuged. The supernatants were then incubated with a precipitation antibody against ubiquitin (Santa Cruz Biotechnology) and protein A/G agarose for 1.5 h at 4°C. Precipitates were collected by centrifugation, washed thrice with wash buffer, and solubilized with 4× Laemmli Sample Buffer (Bio-Rad, Hercules, CA, USA) and 2-mercaptoethanol (Sigma). Finally, immunoblotting was performed using antibodies against GLI2.
5
Chromatin Immunoprecipitation Protocol
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Cells were fixed in 1% formaldehyde for 20 min and then quenched with 0.125 M lysine, followed by swelling in lysis buffer (50 mM Hepes, pH 7.5, 140 mM NaCl, 1% Triton X-100, 0.1% NaDeoxycholate, and protease inhibitors) for 30 min on ice. Chromatin was sheared to an average length of 400 bp by sonication. After being de-cross-linked by RNase, proteinase K, and heat, input genomic DNA was precipitated with ethanol and quantified in a GeneQuant 100 spectrophotometer (GE Healthcare). Chromatin was precleared with protein A/G-agarose (Santa Cruz Biotechnology, Inc.), followed by incubation with the indicated antibodies at 4°C overnight and further incubation with protein A/G-agarose for 2 h. Beads were washed with washing buffer (10 mM Tris, pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% NaDeoxycholate, 1 mM EDTA) three times and eluted with elution buffer (50 mM Tris, pH 8.0, 1% SDS, 10 mM EDTA). Eluates were de-cross-linked by RNase, proteinase K, and heat, and DNA was extracted with phenolchloroform, followed by ethanol precipitation. For each ChIP experiment, 2 × 104 cells were used. 5% of nuclear extracts served as inputs. Immunoprecipitated DNAs were further analyzed by real-time PCR. Signals were normalized to input DNA.
6
Chalcone Derivative and Resveratrol Effects on Osteoblasts
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Mouse osteoblasts were incubated with the chalcone derivative (20 μM) or resveratrol (20 μM) or vehicle (DMSO) for 12 h. Cell lysates were prepared in HEPES lysis buffer (20 mM HEPES pH 7.2, 50 mM NaCl, 0.5% Triton X-100, 1 mM NaF, 1 mM dithiothreitol) supplemented with protease inhibitor cocktail (Roche Life Science) and phosphatase inhibitors (10 mM NaF and 1 mM Na3VO4). Immunoprecipitation was performed using an anti-Smurf1 antibody (1: 50, 45-K, Santa Cruz Biotechnology) and protein A/G-agarose (Santa Cruz Biotechnology) at 4 °C25 (link),49 (link). For mutation studies, mouse osteoblasts were transfected with Flag-Smurf1 or Flag-Smurf1 mutants for 40 h and then incubated with the chalcone derivative (20 μM) or vehicle (DMSO) for 12 h. Cell lysates were prepared and immunoprecipitation was performed using an anti-Flag antibody (5.0 mg/ml, F7425, Sigma) and protein A/G-agarose (Santa Cruz Biotechnology) at 4 °C25 (link),49 (link).
7
Immunodepletion of EGF from Murine Breast Milk
8
VDAC1 Immunoprecipitation and PDZD8 Detection
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Immunoprecipitation was performed according to a previously described method [37 (link)]. Lysates were pre-cleaned in lysis buffer with protein A/G agarose (Santa Cruz Biotechnology) for 1 h at 4 °C and subsequently centrifuged. The supernatants were then incubated with a precipitation antibody against VDAC1 (Santa Cruz) and protein A/G agarose for 1.5 h at 4 °C. Precipitates were collected by centrifugation, washed thrice with wash buffer, and solubilized with 4× Laemmli Sample Buffer (Bio-Rad, Hercules, CA, USA) and 2-mercaptoethanol (Sigma). Finally, immunoblotting was performed using antibody against PDZD8 (Bioss Inc). For secondary antibody, VerBlot for IP Detection Reagent HRP (Abcam) was used.
9
EGFR Immunoprecipitation and Analysis
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Cells were lysed in RIPA buffer with Phosphatase Inhibitor Cocktail (Nacalai tesque). The lysates were precleared with protein A/G-agarose (Santa Cruz Biotechnology) and control IgG at 4 °C for 30 minutes.
The lysates were then incubated with anti-EGFR antibodies (Santa Cruz Biotechnology) at 4 °C, followed by incubation with protein A/G-agarose. The immunoprecipitated proteins were washed with RIPA buffer, and were subjected to electrophoresis.
The lysates were then incubated with anti-EGFR antibodies (Santa Cruz Biotechnology) at 4 °C, followed by incubation with protein A/G-agarose. The immunoprecipitated proteins were washed with RIPA buffer, and were subjected to electrophoresis.
10
Immunodepletion of EGF from Murine Breast Milk
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Immunodepletion of EGF from breast milk was performed as in 21 (link). Briefly, 500ul aliquots of freshly harvested murine breast milk were filtered via Amicon Ultra 10K Centrifugal Filters (Millipore) at 13,000 RPM for 15 minutes. Filtered aliquots were then incubated at 4 degrees Celsius for 30 minutes with control goat IgG antibody (10 μl) and protein A/G-agarose (20 μl, Santa Cruz Biotechnology), and centrifuged at 3000RPM for 30 seconds. The supernatant was incubated for 1 h at 4 degrees Celsius with 2μg of anti-EGF antibody (Santa Cruz Biotechnology) followed by overnight incubation on a rotator with an additional 20 μl of protein A/G-agarose. Supernatants were collected after centrifugation at 3000RPM for 30 seconds and an EGF ELISA (Abcam) was performed as per manufacturers instructions using a 1:500 dilution (wild-type murine breast milk [22907±976 pg/ml]; EGF-depleted murine breast milk 468.5±87.1 pg/ml].
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