Immunoprecipitation was carried out using HCC1937 and MDA‐MB231 cells expressing Halo fusion proteins. The cell lysates were incubated with each antibody and then TrueBlot anti‐rabbit Ig IP beads (Rockland, Limerick, PA, USA) were added. After incubation, the binding proteins were eluted from the beads with 1× SDS Sample Buffer (62.5 mmol/L Tris pH 6.8, 2% SDS, 10% glycerol, 0.1 mol/L DTT, 0.005% bromophenol blue, 2% 2‐mercaptoethanol). The following antibodies were used for IP: HSP90α (ab2928), and GRP78 (ab21685) from Abcam Inc. (Cambridge, MA, USA), and γ‐catenin (#2309) from Cell Signaling Technology Inc. (Beverly, MA, USA).
Trueblot anti rabbit ig ip beads
Manufactured by Rockland Immunochemicals
Sourced in United States
TrueBlot anti-Rabbit Ig IP Beads are a protein G-based immunoprecipitation reagent designed for the selective capture of rabbit immunoglobulins from biological samples. The beads provide an efficient way to isolate and concentrate target proteins for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Mouse yap, by Santa Cruz Biotechnology (2 mentions)
True blot hrp conjugated anti rabbit igg, by Rockland Immunochemicals (2 mentions)
Mouse tead1, by BD (2 mentions)
Tris glycine precast gel, by Bio-Rad (2 mentions)
True blot hrp conjugated anti mouse igg, by Rockland Immunochemicals (2 mentions)
Protein a g magnetic bead, by Thermo Fisher Scientific (2 mentions)
Hsp90α ab2928, by Abcam (1 mentions)
γ catenin 2309, by Cell Signaling Technology (1 mentions)
2 mercaptoethanol, by Bio-Rad (1 mentions)
Sds sample loading buffer, by Bio-Rad (1 mentions)
Ecl detection reagent, by Thermo Fisher Scientific (1 mentions)
Protein g sepharose, by GE Healthcare (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
Protein a sepharose, by Repligen (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
6 protocols using trueblot anti rabbit ig ip beads
1
Immunoprecipitation of Halo Fusion Proteins
2
IP of HA and NCL Proteins
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Cell lysates were incubated with anti-HA and anti-NCL antibodies at 4 °C overnight with gentle rocking. Then, the samples were incubated with TrueBlot anti-rabbit Ig IP beads (Rockland, Gilbertsville, PA, USA, Cat. No. 88-1688-31) at 4 °C for 4 h with gentle rocking before being boiled for 5 min in SDS sample loading buffer (Bio-Rad, Hercules, CA, USA, Cat. No. 1610747) containing 2-mercaptoethanol (Bio-Rad, Cat. No. 1610710).
3
Immunoprecipitation of YAP and TEAD1
4
Immunoprecipitation of YAP and TEAD1
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Retinae were homogenized in lysis buffer (20mM Tris-HCl [pH 7.5], 150mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton-X, 2.5mM sodium pyrophosphate, 1mM βglycerophosphate, 1mM Na3VO4). Aliquots of clarified protein lysate (500mg per sample) were diluted in 500μL lysis buffer supplemented with HaltTM Protease Inhibitor Cocktail (ThermoFisher, #78430) and incubated with 1μg of the following antibodies overnight at 4°C: mouse-YAP (Santa Cruz, 101199 (Zanconato et al., 2015 (link))) or mouse-TEAD1 (BD Transduction, 610923, (Zanconato et al., 2015 (link))), Rabbit IgG (Millipore, #12–370), Mouse IgG (Millipore, #12–371) overnight at 4°C (Poché et al., 2016 (link)). The protein/antibody complexes were captured using either Protein A/G Magnetic Beads (ThermoFisher, #88802) or TrueBlot anti-Rabbit Ig IP Beads (Rockland, #00–8800-25) by incubating for 1 hour at room temperature. The beads were then washed, and protein eluted with SDS-PAGE reducing sample buffer (50μL 1X PBS plus 10μL 6X sample buffer) by boiling for 10 minutes. Next, 2% of input protein and 15μL of IP reactions were loaded onto a 4%–15% Tris/Glycine precast gels (BioRad, 5671084) and western blots performed as described. The following secondary antibodies were used: True Blot HRP-conjugated anti-Mouse IgG (1:1000, Rockland, #18–8817-33) and True Blot HRP-conjugated anti-Rabbit IgG (1:1000, Rockland, #18–8816-33).
5
Western Blotting and Immunoprecipitation Protocol
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Western blotting was performed using standard protocol. Briefly, cells were lysed with 1% SDS lysis buffer (50 mM Tris pH 6.8, 1% SDS) and protein concentration was determined using BCA Assay Kit (Thermo Fisher). Samples were resolved by SDS-PAGE, transferred to PVDF membranes and blotted with the desired antibodies. Protein expression was detected by ECL Detection Reagent (Thermo Fisher).
For immunoprecipitation, cells were rinsed twice with ice-cold PBS and lysed in ice-cold mild lysis buffer (10 mM Tris pH 7.5, 10 mM EDTA, 100 mM NaCl, 1% NP40, 50 mM NaF, 1 mM Na 3 VO 4 , 0.1 mM DTT) supplemented with EDTA-free Complete Protease Inhibitor. The soluble fractions of cell lysates were isolated by centrifugation at 14,000 g for 10 min at 4 C. Primary antibodies were added to lysates and incubated with rotation for 2 hr at 4 C. 10 ml of a 50% slurry of protein G-Sepharose (GE healthcare) for mouse or protein A-Sepharose (Repligen Corporation) for rabbit was added and the incubation continued for an additional 1.5 hr. TrueBlot Anti-Rabbit Ig IP Beads (Rockland Immunochemicals) were used for immunoprecipitation of endogenous AMPKg1 and eEF2. Immunoprecipitates were washed five times with lysis buffer. Immunoprecipitated proteins were denatured by the addition of 80 ml sample buffer and boiling for 5 min.
For immunoprecipitation, cells were rinsed twice with ice-cold PBS and lysed in ice-cold mild lysis buffer (10 mM Tris pH 7.5, 10 mM EDTA, 100 mM NaCl, 1% NP40, 50 mM NaF, 1 mM Na 3 VO 4 , 0.1 mM DTT) supplemented with EDTA-free Complete Protease Inhibitor. The soluble fractions of cell lysates were isolated by centrifugation at 14,000 g for 10 min at 4 C. Primary antibodies were added to lysates and incubated with rotation for 2 hr at 4 C. 10 ml of a 50% slurry of protein G-Sepharose (GE healthcare) for mouse or protein A-Sepharose (Repligen Corporation) for rabbit was added and the incubation continued for an additional 1.5 hr. TrueBlot Anti-Rabbit Ig IP Beads (Rockland Immunochemicals) were used for immunoprecipitation of endogenous AMPKg1 and eEF2. Immunoprecipitates were washed five times with lysis buffer. Immunoprecipitated proteins were denatured by the addition of 80 ml sample buffer and boiling for 5 min.
6
Immunoprecipitation and Immunoblotting of Endogenous MCM8
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To detect endogenous MCM8 in RPE1-hTERT-derived cell lines, we performed MCM8 immunoprecipitation/immunoblotting using the TrueBlot system (Rockland). Cells (4 × 106) were lysed in 400 µL of MCM8 IP buffer (50 mM HEPES at pH 7.4, 100 mM KCl, 0.5% NP-40, 2 mM EDTA, 10 mM NaF, Complete protease inhibitor cocktail [Roche]) for 30 min on ice and cleared by centrifugation. Five microliters of rabbit anti-MCM8 antibody was added, and samples were incubated for 2 h at 4°C with rotation. Fifty microliters of pre-equilibrated TrueBlot antirabbit Ig IP beads (Rockland) was added to each sample prior to 1 h of rotation at 4°C. Beads were washed three times with MCM8 IP buffer and boiled in SDS sample buffer, and the supernatant was used for immunoblotting with rabbit anti-MCM8 and horseradish peroxidase-conjugated rabbit IgG TrueBlot (Rockland) as primary and secondary antibodies, respectively.
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