DG of 3-week-old WT mice were harvested and homogenized in 1ml of homogenization buffer (25mM Tris [pH 7.0], 25mM Tris [pH 8.0], 100mM KCl, 12mM MgCl2, 10% IGEPAL, 1mM DTT, Protease inhibitors, 1x RNase inhibitor, 200 units/mL, 100μg/mL Cycloheximide, 1mg/mL Heparin) with 2X complete protease inhibitors (Boehringer-Mannheim). Nuclei and debris were pelleted at 14,000 X g for 15 min. The resulting supernatant was pre-cleared for 1 h with 100 μl recombinant protein G agarose (Invitrogen) (washed with lysis buffer first). An aliquot of pre-cleared input was saved for RNA extraction (20 μl). A monoclonal antibody against Fmr1 (7F1-1-C, DSHB) was incubated with supernatant at 4ºC for 4 h and before add Dynabeads (lifetech). The antibody/FMRP conjugation was rotated at 4ºC overnight. After third wash with the High salt buffer (25mM Tris [pH 7.0], 25mM Tris [pH 8.0], 300mM KCl, 12mM MgCl2, 1mM DTT, 100μg/mL Cycloheximide), the immunoprecipitations was re-suspended into Trizol (Invitrogen) for RNA isolation. RNA from IP and input was used and all Real-time PCR reactions were carried out in duplicate for each sample on each amplicon. The relative expression levels of genes in specific Fmr1-immunoprecipitations compared with non-specific immunoprecipitations (mouse IgG only) was calculated.
Recombinant protein g agarose
Manufactured by Thermo Fisher Scientific
Sourced in United States
Recombinant Protein G Agarose is a laboratory product used for the purification of antibodies from various sample types. It consists of the bacterial protein G immobilized on an agarose resin matrix. Protein G has a high affinity for the Fc region of immunoglobulins, enabling the capture and isolation of antibodies from solution.
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Lab products found in correlation
Amicon ultracentrifuge filter device, by Merck Group (4 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
T4 ligase, by New England Biolabs (3 mentions)
Complete protease inhibitor, by Roche (2 mentions)
Dynabead, by Thermo Fisher Scientific (2 mentions)
Mabe14, by Merck Group (2 mentions)
Laemmli electrophoresis sample buffer, by Bio-Rad (2 mentions)
Normal mouse or rabbit igg, by Santa Cruz Biotechnology (2 mentions)
Anti flag m2 agarose, by Merck Group (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Nuclei ez lysis buffer, by Merck Group (2 mentions)
Lysis buffer, by Cell Signaling Technology (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Rnase inhibitor, by Roche (1 mentions)
Nonidet p 40, by Roche (1 mentions)
Anti myc 9e10 sc 40, by Santa Cruz Biotechnology (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Phosstop, by Roche (1 mentions)
Anti phb1 antibody, by Santa Cruz Biotechnology (1 mentions)
25 protocols using recombinant protein g agarose
1
Immunoprecipitation of FMRP from Mouse Brain
2
Immunoprecipitation of Dystrophin Isoforms
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Immunoprecipitation of Dp71d (20 μl of Dys-2 Ab) and Dp71f (20 μl of 5F3 Ab) or both dystrophin isoforms (20 μl of Mandra-1 Ab) was performed with the nucleoskeleton fraction from cultured hippocampal neurons. Briefly, nucleoskeleton fraction (1 mg) was precleared with 20 μl of recombinant protein G-Agarose beads overnight at 4°C. The beads were removed by centrifugation at 16,000 rpm for 5 min, and precleared extract was incubated overnight at 4°C with the immunoprecipitating Ab, previously bound to recombinant protein G-Agarose (Invitrogen, Carlsbad, CA, USA). As negative control, parallel incubation with a non-related Ab (monoclonal anti-CD4 Ab, as nonspecific immunoprecipitation) was performed. The immune complexes were collected by centrifugation at 16,000 g for 5 min, and washed three times for 10 min with 1 ml of RIPA buffer and then eluted by boiling in Laemmli buffer. Immunoprecipitated proteins were then analyzed by western blot as described above.
3
Immunoprecipitation and Telomerase Assay
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Plasmid pCl neo‐hEST2 together with TERC (pBS U3‐hTR‐500) were transfected into log phase Hek293 cells and non‐denaturing cellular lysates were prepared 48 h later in lysis buffer (Cell Signaling Technology, Beverly, MA). Immunoprecipitation was performed as described previously.21 Briefly, transfected cells were harvested, washed in PBS, lysed in cell lysis buffer, and clarified by cold centrifugation (14 000× g for 10 min). An aliquot of supernatant containing 500 µg protein was incubated with 2 µg anti‐hTERT antibody MABE14 (EMD Millipore, MA) at 4ºC overnight with gentle mixing. Then, 20 µl of recombinant Protein G Agarose (Invitrogen, CA) was added and incubated at 4ºC for 3 h. IPs were collected by centrifugation at 3,000 rpm for 30 s at 4ºC, washed three times with ice‐cold PBS, aliquoted, and stored at −80°C until use. For denaturing gel electrophoresis, aliquots were dissolved in 2X Laemmli electrophoresis sample buffer (Bio‐Rad, CA) and assayed by western blot (WB). Normal rabbit or mouse IgG was always used as a control (Santa Cruz, CA). Aliquots of the IP were also assayed in triplicate by TRAP assay and quantified using realtime PCR as described above. In some cases, aliquots were electrophoresed on non‐denaturing agarose gels after treatment with MPP and/or nucleases.
4
Immunoprecipitation of GFP-Fusion Proteins
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Cell lysates (800 μg total protein in a volume of 400 μl lysis buffer; see above) were pre-cleared with recombinant Protein G Agarose (10 μl, cat #15920-010, Invitrogen). Anti-GFP saturated rProtein G Agarose was prepared by incubating 50/50 rProtein G Agarose/TBS slurry (800 μl) with anti-GFP (56 μg) and incubated for 2 hours on a rotation platform at 4°C. The precleared extracts were then incubated with the anti-GFP saturated rProtein G-Agarose on a rotating platform o/n at 4°C. The beads were vigorously washed three times with ice-cold lysis buffer. 2× loading buffer (30 μl; 125 mM Tris–HCl pH 6.8, 4% (w/v) SDS, 20% (w/v) Glycerol and 0.004% (w/v) BromPhenol Blue) was added to the beads and the samples were incubated at 95°C for 5 minutes. Proteins were resolved by 8% SDS-PAGE and subjected to immunoblotting.
5
Immunoprecipitation of HCV NS3 and hTERT
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Immunoprecipitation was performed as described previously [20 (link)], with minor modifications. Briefly, log-phase HCV infected cells, Huh5-15NS replicons, or vector transfected HEK-293 cells were harvested, washed in PBS, lysed in cell lysis buffer (Cell Signaling Technology, Beverly, MA) and clarified by cold centrifugation (14,000xg for 10 min). An aliquot of supernatant containing about 500 μg protein was incubated with 2μg anti-NS3 monoclonal antibody (Meridian Life Science, Saco, ME) or anti-hTERT antibody MABE14 (EMD Millipore, MA) at 4°C overnight with gentle mixing. Then, 20 μl of recombinant Protein G Agarose (Invitrogen, CA) was added and incubated at 4°C for 3hrs. Immunoprecipitates were collected by centrifugation at 3,000 rpm for 30s at 4°C, washed three times with ice-cold PBS, then dissolved in 40μl 2x Laemmli electrophoresis sample buffer (Bio-Rad, CA) and assayed by WB. Normal rabbit or mouse IgG was always used as control (Santa Cruz, CA). In some cases, cell lysates were incubated with RNAases A or H (Life Technologies, NY); (50 or 100 μ/ml, 37°C, 20 min and 0.2 u/ml 37°C, 20 min, respectively) or DNAase 1, (Qiagen, CA) (70 μ/ml at room temperature, 1 hr), or ethidium bromide (0.1 mg/ml at room temperature, 1 hr) prior to immunoprecipitation.
6
FMRP Interactome Identification via RNA-IP
7
Immunoprecipitation of FMRP from Mouse Brain
8
Purification of Total IgG from Human Serum
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Human serum was obtained through centrifuging healthy adult donors' peripheral blood at 500 × g for 5minutes at 4°C. Total IgG was purified from serum using affinity chromatography with Recombinant Protein G Agarose according to the manufacturer's instructions (Invitrogen, California, USA).
9
Immunoprecipitation of BRCA2 using Anti-BRCA2 Antibody
10
Immunoprecipitation Assay for Protein Interactions
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For immunoprecipitation (IP) assay, transfected HEK 293T cells were lysed in lysis buffer (50 mM Tris-HCl (pH7.4) containing 1.0% Nonidet P-40, 150 Mm NaCl, 5 mM EDTA, protease inhibitor (04693132001, Roche) and phosphatase inhibitor (PhosStop, 04906837001, Roche), then spun and the supernatant was collected for IP. IP analysis was performed by incubating the supernatant with anti-Myc (9E10) (sc-40, Santa Cruz) or anti-FLAG M2 (F1804, Sigma-Aldrich, Missouri, USA) antibodies at 4°C for 1 hr. Then recombinant Protein G Agarose (15920–010, Invitrogen, Hong Kong) was added into the mixture and incubated at 4°C for 1 hr. After incubation, rProtein G Agarose was washed with lysis buffer three times. The precipitated proteins were collected by boiling the rProtein G Agarose with loading buffer.
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