Prp16p (3 μg) and Sec63-2p (1.5 μg) were incubated overnight at 4°C with protein A dynabeads (Roche) and anti-Prp16p-029 (Eurogentec, this study) at 1/100 dilution in IP buffer (50-mM Tris–HCl, pH 7.5, 150-mM NaCl, 1-mM MgSO4, 0.02% (v/v) NP-40) containing bovine serum albumin (BSA) (100 μg/ml), glycogen (50 μg/ml) and tRNA (100 μg/ml). For co-immunoprecipitations treated with RNase A (30 ng/μl), tRNA was omitted. After extensive washing with IP buffer, beads were loaded onto NuPAGE 4–12% gradient gels (Invitrogen), followed by western blotting using anti-His-HRP-conjugated antibodies (sc-8036, Santa Cruz; 1/3000 dilution).
Protein a dynabeads
Manufactured by Roche
Sourced in United States
Protein A Dynabeads are superparamagnetic beads coated with recombinant Protein A, a bacterial protein that binds to the Fc region of immunoglobulins. They are designed for the rapid and efficient purification of antibodies from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Sc 8036, by Santa Cruz Biotechnology (1 mentions)
Nupage 4 12 gradient gel, by Thermo Fisher Scientific (1 mentions)
Complete proteinase inhibitor, by Roche (1 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (1 mentions)
Ab6556, by Abcam (1 mentions)
H6908, by Merck Group (1 mentions)
Na934, by GE Healthcare (1 mentions)
α gfp antibody, by Roche (1 mentions)
Cfx96 real time system c1000 thermal cycler, by Bio-Rad (1 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (1 mentions)
Proteinase k, by Roche (1 mentions)
4 protocols using protein a dynabeads
1
Co-immunoprecipitation of Prp16p and Sec63-2p
2
Immunoprecipitation Protocol for Protein Analysis
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Precleared the cell lysates (500 μg of total protein) with 1.5 mg of protein A Dynabeads (Life Technologies, Grand Island, NY, USA) for 1 h at 4°C, then incubated with the appropriate antibodies bound to protein A Dynabeads for 1 h at 4°C and washed three times with lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, supplemented with complete proteinase inhibitor; Roche Applied Sciences). The beads were separated on a magnet and the immunocomplexes were resuspended in 20 μL of SDS-PAGE sample buffer for further analysis [13 (link)].
3
Co-IP and ChIP Assays for DELLA Protein Interactions
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For Co-IP assays, the p35S:MtDELLA1-GFP, p35S: MtDELLA2-GFP and p35S: MtDELLA3-GFP translational fusions were introduced into an A. tumefaciens AGL-0 strain and the p35S:HA-NSP1 (ref. 16 (link)), p35S:HA-NSP2 (ref. 16 (link)) and p35S:HA-NF-YA1 (ref. 15 (link)) translational fusions into an A. tumefaciens GV3103 strain. After co-transformation in N. benthamiana leaves, Co-IPs were performed using Invitrogen Protein A Dynabeads coupled with a α-GFP antibody (Roche 11814460001)71 (link). Proteins were separated by SDS–polyacrylamide gel electrophoresis and detected by immunoblotting using a α-GFP antibody (AbCam Ab3277; 1/2,000) or a α-HA antibody (Sigma H6908; 1/5,000) and a α-rabbit horseradish peroxidase secondary antibody (GE Healthcare NA934; 1/10,000). Full blots are shown in Supplementary Fig. 8 . For ChIP assays, M. truncatula roots transformed with the pMtDELLA1:GFP:della1-Δ18 (ref. 32 (link)) construct (or GFP as a negative control) were analysed one dpi with S. meliloti. IP were performed using a α-GFP antibody (AbCam Ab6556; 1/200), and PCR with primers shown in the Supplementary Table 5 .
4
DNA Extraction and DRIP Assay
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5x106 cells were collected, washed in PBS and resuspended in 1.6 mL of TRIS-EDTA buffer pH8.0. Cells were lysed by addition 50 μL SDS 20% and 5 μL ProteinaseK 20 mg/ml (Roche), mix gently and incubated overnight at 37°C. DNA was extracted with phenol/chloroform in phase lock tubes, precipitated with EtOH/sodium acetate, washed three times with 70% EtOH, and resuspended in TE. DNA was digested with EcoRI, HindIII, BsrGI, SspI and XbaI (NEB) restriction enzymes overnight at 37°C and DNA was isolated as described above. For DRIP 4.4 μg of digested DNA was diluted in 500 μL TE buffer pH8.0, 50 μL was kept as input and 50 μL of 10x binding buffer (100 mM NPO4 pH7.0, 1.4 M NaCl, 0.5% Triton X-100) and 10 μL of S9.6 antibody was added to the rest and incubated overnight at 37°C. Protein A Dynabeads (Roche, 10002D) were added for 2 h. Bound beads were washed 3 times in binding buffer 1x and elution was performed in elution buffer (50 mM Tris pH 8, 10 mM EDTA, 0.5% SDS, Proteinase K) for 45 min at 55°C. DNA was purified as described and resuspended in 10 mM TrisHCl, pH 8.0. Quantitative PCR of immunoprecipitated DNA fragments was performed on Bio-Rad CFX96 Real-Time System C1000 Thermal Cycler using SsoAdvanced Universal SYBR® Green Supermix (Bio-Rad, 1725271).
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