Cells were grown to confluence in 10 cm plates. Plates were washed twice on ice in ice-cold PBS and then lysis buffer was added with a protease inhibitor cocktail (Roche). Lysates collected after 10 min and pre-cleared by centrifugation at 13,000 G for 10 min at 4 °C. The supernatant was removed from the debris pellet and transferred to a clean tube. Protein concentration was calculated using the Bradford Protein Assay system (Bio-Rad). Fifty microlitres of Dynabeads (Thermo) were prepared according to manufacturer's instructions. Antibodies (2–4 μg) were bound to the beads by incubation at room temperature with rotation for 30 min. Species matched IgG (Rabbit IgG sc-2027, Mouse IgG sc-2025, Santa Cruz) were bound to beads as a control. Two milligram of protein lysate was added to beads and incubated overnight at 4 °C with rotation. The following day, beads were washed three times for 5 min on ice in 700 μl NP-40 wash buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA and 1% NP40) containing protease and phosphatase inhibitors (Roche) followed by separation on a magnet. Beads were separated from antibody–protein complexes by boiling for 5 min in 2 × Laemelli buffer and the eluate separated by a magnet loaded onto a gel for SDS–PAGE electrophoresis. The lysate was run alongside as an input for the immunoprecipitation.
Mouse igg sc 2025
Manufactured by Santa Cruz Biotechnology
Sourced in United States, Germany
Mouse IgG (sc-2025) is a purified immunoglobulin G (IgG) fraction from mouse serum. It can be used as an isotype control for immunological experiments.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit igg sc 2027, by Santa Cruz Biotechnology (5 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Ab2912, by Abcam (2 mentions)
Ab11425, by Abcam (2 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (2 mentions)
Quick start bradford dye reagent, by Bio-Rad (2 mentions)
Bis tris gel, by Bio-Rad (2 mentions)
Dynabeads protein g, by Santa Cruz Biotechnology (2 mentions)
Sc10646, by Santa Cruz Biotechnology (2 mentions)
Ac003, by Alomone (2 mentions)
Ac062, by Alomone (2 mentions)
Goat igg sc2028, by Santa Cruz Biotechnology (2 mentions)
Bradford protein assay system, by Bio-Rad (1 mentions)
Dynabead, by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Protein g a plus agarose beads, by Santa Cruz Biotechnology (1 mentions)
Direct zol rna microprep kit, by Zymo Research (1 mentions)
β actin antibody a1978, by Merck Group (1 mentions)
Dichloroacetate dca, by Merck Group (1 mentions)
Dinaciclib hy 10492, by MedChemExpress (1 mentions)
14 protocols using mouse igg sc 2025
1
Immunoprecipitation Protocol for Protein Complexes
2
Immunoprecipitation of Cardiac NCX1 Protein
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Left ventricle lysate was incubated with the specific antibodies (2 μg/IP), 50 μl protein PLUS A/G agarose beads (sc-2003, Santa Cruz) and with or without presence of 100 μM NOPT-TAT or OPT overnight at 4°C. Immunocomplexes were washed twice in IP buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton), centrifuged at 3,000 g for 1 min at 4°C and boiled in SDS loading buffer before the immunoblot analyses were undertaken. Equal amount of mouse IgG (sc-2025, Santa Cruz) or a custom made anti-NCX1 blocking peptide (CGQPVFRKVHARDHPIPST) incubated with anti-NCX1 prior to immunoprecipitation (Hafver et al., 2016 (link)) was used as negative control.
3
RNA Immunoprecipitation and RT-qPCR Analysis
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We performed RNA immunoprecipitation following published protocols 77 (link). Briefly, cells were lysed in an ice cold polysome lysis buffer with 3–5 million cells per 1 ml of lysis buffer. The lysate was spun down at 16000xg for 15 minutes at 4°C, and the supernatant was transferred to a new tube. 50µl of equilibrated protein A/G-agaraose beads (Pierce, Thermo Fisher) were added to the supernatant for preclearing at 4°C for 1h. 100µl of the lysate was saved as the input sample, and the rest was incubated with 5µg of eIF4E antibody (sc-271480, Santa Cruz) or Mouse IgG (sc-2025, Santa Cruz) at 4°C overnight. The next day, 50µl of equilibrated protein A/G-agaraose beads were added to each sample and rotated at 4°C for 4 hours. Then, 100µl of the supernatant was saved for the flow-through sample, and the beads were washed with lysis buffer 2 times, and then washed with lysis buffer containing 1M urea another 2 times. Then, the beads were boiled in Tris-EDTA containing 1% SDS and 12% β-Mercaptoethanol before RNA was extracted using a Direct-zol RNA Microprep kit (Zymo Research). The extracted RNA was then prepared for RT-qPCR analysis to examine RB1, Actin, and GAPDH expression.
4
Cardiac Protein Immunoprecipitation and Analysis
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The study was carried out following the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health and the Directive 2010/63/EU of the European Parliament. 6-8 weeks old male mice of strain C57BL6 were sacrificed by cervical dislocation and their hearts were harvested and snap frozen in liquid nitrogen and stored at -80 °C. Heart tissue was homogenized on a Precellys 24 and solubilized in ice-cold lysis buffer containing protease and phosphatase inhibitors. Tissue lysates were centrifuged to remove insoluble debris. For each tissue preparation produced, lysates derived from 5 mice were pooled and protein concentrations were measured by Quick Start Bradford Dye Reagent (Biorad). Solubilized heart tissue lysate was pre-cleared with Dynabeads protein G (Invitrogen) before incubation with primary antibody followed by binding to Dynabeads protein G, using either anti-KCNQ1 (10 μl SC10646, Santa Cruz), anti-CACNA1C (2 μl AC003, Alomone), anti-KCNH2 (2 μl AC062, Alomone), anti-CAV3 (2 μl ab2912, Abcam), anti-SNTA1 (2 μl ab11425, Abcam) or control IgG (1.5 μl goat IgG: SC2028, 1.5 μl rabbit IgG: SC2027, 1.5 μl mouse IgG: SC2025, Santa Cruz). After washing, bound proteins were eluted with 1× sample buffer containing 100 mM dithiothreitol (70 °C, 3 min) and separated by SDS-PAGE (4-15 % Bis-Tris gels, BioRad).
5
Antibody and Inhibitor Protocols for Cell Research
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The antibody for FOXK2 (ab5298) was from Abcam Biotechnology company (Cambridge, United Kingdom). The β-Actin antibody (A1978) was from Sigma (St. Louis, MO) and mouse IgG (sc-2025) were from Santa Cruz Biotechnology (Dallas, TX). Doxorubicin (Dox, D1515), etoposide (VP16, E1383), 5-Fluorouracil (5FU, F6627), and Dichloroacetate (DCA) (347795) were from Sigma (St. Louis, MO). CDK2 inhibitors, Dinaciclib (HY-10492) and Alpelisib (BYL-719, HY-15244), were from MedChemExpress (Monmouth Junction, NJ).
6
Antibody Procurement for Cellular Analysis
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Anti-HIF-1α (610958) and anti-CPE (610758) antibodies were purchased from BD Biosciences (USA). Anti-CPA4 (ab81543) and anti-14-3-3γ (MAB5700) antibodies were obtained from Abcam (USA) and R&D Systems (USA), respectively. Mouse IgG (sc-2025) and anti-H3K4me3 (9751S) were obtained from Santa Cruz Biotechnology (USA) and Cell Signaling Technology (USA), respectively. Ponceau S solution and Oil Red O stain were supplied by Sigma-Aldrich (USA).
7
Antibody Optimization for Western Blot Analysis
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For Western blot, primary antibody catalog numbers and dilution factors are as follows: polyclonal anti–BNIP-2 antibodies were purchased from Sigma-Aldrich (HPA026843) and GeneTex (GTX114283) or self-purified from rabbit serum, as previously described (28 (link)); monoclonal anti–GEF-H1 (ab155785) was purchased from Abcam; monoclonal anti-RhoA was from Santa Cruz Biotechnology (sc-418); polyclonal anti–Phospho-Myosin Light Chain 2 (Ser19) antibody was from Cell Signaling Technology (#3671); monoclonal anti–α-tubulin was from Sigma-Aldrich (T9026); anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was from Life Technologies (Invitrogen); rabbit immunoglobulin G (IgG; sc-2027); and mouse IgG (sc-2025) were from Santa Cruz Biotechnology. The horseradish peroxidase secondary antibodies polyclonal antibody against FLAG and polyclonal antibody against HA were from Sigma-Aldrich. For immunostainings, all secondary antibodies conjugated with Alexa Fluor dyes and Alexa Fluor Phalloidin were from Life Technologies. For inhibitor treatment, Rho inhibitor I (catalog no. CT04) and Rho activator II (catalog no. CN03) were from Cytoskeleton Inc. DMSO, Y-27632, blebbistatin, and nocodazole were from Sigma-Aldrich.
8
Immunoblotting Antibody Protocol
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Anti-TH (T-1299) and anti-β-tubulin from Sigma-Aldrich. Anti-Ub (sc-8017), rabbit IgG (sc-2027), and mouse IgG (sc-2025) from Santa Cruz Biotechnology (Santa Cruz, CA); Anti-pSer PKC motif (#2261) and anti-cleaved caspase3 (#9661) from Cell Signaling (Danvers, MA); anti-CSPα (ab90499) and anti-SNAP25 (ab41455) from Abcam (Cambridge, UK); anti-SNAP25 (MAB331) from Millipore (Billerica, MA); Horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse secondary antibodies from Jackson ImmunoResearch Inc. (West Grove, PA). It is noted that anti-CSPα (ab90499) and anti-SNAP25 (ab41455) antibodies were raised in rabbit and Anti-TH (T-1299) and anti-SNAP25 (MAB331) antibodies were raised in mouse.
9
Immunoprecipitation and Western Blotting of p53, DNAJA1, and CHIP
10
Antibody Characterization Protocol
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Anti-BCCIP (16043-1-AP) antibody was obtained from Proteintech Group (China, Wuhan). Anti-YY1 (sc-1703X) antibody, rabbit IgG (sc-2027) and mouse IgG (sc-2025) were purchased from Santa Cruz Biotechnology (U.S.A.). Anti-Flag (M2)-Agarose and monoclonal antibody (F3165) were from Sigma (U.S.A.). Anti-INO80 (NM_017553, residue 1–526 aa), anti-Arp8 (NM_022899, full length), and anti-GAPDH (NM_002046, full length) rabbit polyclonal antibodies were raised against bacterially expressed proteins (Jilin University).
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