Mouse brains or ER microsomes fraction of mouse brains were homogenized in IP buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 10% glycerol, 50 mM NaF,10 mM glycerolphosphate, 2 mM EGTA, 2 mM EDTA, 1% NP-40, and 1× Protease and Phosphatase Inhibitor Cocktails) with Dounce homogenizer (10 strokes)54 (link). Lysates were centrifuged at 15,000 × g for 15 min at 4 °C, and the supernatants were collected. The protein concentration of the lysates was measured and adjusted to 1 mg/ml. After pre-clearing with Protein G agarose (Thermo Fisher Scientific), the lysates were incubated with antibody-bound Protein G agarose for 1 h at 4 °C. After five washes of the agarose beads with IP buffer at 4 °C, the immune complexes were eluted with SDS sample buffer (Thermo Fisher Scientific) and examined by western blotting. The mouse-derived specific antibody against p150Glued (BD Biosciences, #610474, 1:1000, recognizing p150Glued but not p135+) and normal mouse IgG (Santa Cruz, #sc-2025) were used for co-IP.
Protein g agarose
Manufactured by Thermo Fisher Scientific
Sourced in United States
Protein G agarose is a chromatography resin used for the purification of antibodies. It consists of the bacterial protein G immobilized on an agarose matrix. Protein G has a high affinity for the Fc region of most immunoglobulin classes, allowing the capture and purification of antibodies from complex samples.
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Lab products found in correlation
Protease inhibitor cocktail, by Roche (8 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (7 mentions)
Polyethylenimine (pei), by Polysciences (6 mentions)
Cycloheximide (chx), by Merck Group (4 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (4 mentions)
Halt protease inhibitor, by Thermo Fisher Scientific (4 mentions)
Opti mem, by Thermo Fisher Scientific (4 mentions)
Anti flag m2, by Merck Group (4 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Mg132, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Protein a agarose, by Thermo Fisher Scientific (3 mentions)
Amicon ultra centrifugal unit, by Merck Group (3 mentions)
Normal mouse igg, by Santa Cruz Biotechnology (3 mentions)
Anti flag m2 affinity gel, by Merck Group (3 mentions)
Lipofectamine plus reagent, by Thermo Fisher Scientific (3 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (3 mentions)
Ab17721 and ab90966, by Abcam (2 mentions)
Ab4812, by Abcam (2 mentions)
128 protocols using protein g agarose
1
Immunoprecipitation of p150Glued Protein
2
Co-immunoprecipitation of Protein Interactions
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Cell transfection was performed with Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer's protocol. Briefly, 10 µg of plasmids were mixed with 30 µl Lipofectamine 2000 and incubated at RT for 20 min before adding to the cell cultures. Transfected 293T cells were cultured for another 24 h. Cells were collected and lysed in lysis buffer. The lysates were centrifuged at 15,000×g to remove debris, and the supernatants were incubated with Protein G agarose (Invitrogen, CA, USA) and 2 µg mouse anti-FLAG antibody (Beyotime, Suzhou, China) overnight at 4°C. After a brief centrifugation, the immunocomplexes were washed three times with PBS and subjected to SDS-PAGE. Protein bands were detected by Western blotting with the anti-GFP antibody. A reciprocal co-immunoprecipitation assay was conducted by adding Protein G agarose (Invitrogen, CA, USA) and 2 µg mouse anti-GFP antibody (Beyotime, Suzhou, China) to cell protein extracts overnight at 4°C. After a brief centrifugation, the immunocomplexes were washed three times in PBS and subjected to SDS-PAGE. The proteins were transferred to Western blots, and probed with the anti-FLAG antibody.
3
Antibody-Based Protein Detection Assay
4
Antibody-Based Protein Detection Assay
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Antibodies against human KDM1A (ab17721 and ab90966), CK1δ (ab48031), GATA6 (ab22600), and USP22 (ab4812) were from Abcam. Antibodies against GST (sc-138), Tuj-1(sc-58888), CK1α (sc-6477), CK1ε (sc-6471), BMP2 (sc-6895), CDKN1A (sc-397), and α-Tubulin (sc-5286) were from Santa Cruz Biotechnology. Antibodies against human GSK3β (9315), GSK3α (9338), CD133 (3663), phospho-GSK3β-S9 (9336), Oct4 (2750), H3K4me2/3 (9725 and 9751) and Myc-tag (2276) were from Cell Signaling Technology. Antibodies against phospho-serine/threonine (612548) and nestin (611658) were from BD Transduction Laboratories. Antibodies against hemagglutinin (HA)-Tag and Flag-tag were from Sigma. Hoechst 33342, Alexa Fluor 488 goat anti-rabbit antibody, and Alexa Fluor 594 goat anti-mouse antibody were from Molecular Probes. The specific antibody against phospho-KDM1A S683 was produced by Signalway Antibody. Supplementary Table 1 contains detailed information about the used antibodies.
Temozolomide (TMZ), tideglusib, lithium chloride, glutathione S-transferase (GST) beads, cycloheximide (CHX), and MG132 were from Sigma. Puromycin, hygromycin, and D4476 (4-[4-(2,3-dihydro-benzo[1,4]dioxin-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl] benzamide) were from EMD Biosciences. Protein G agarose was from Life Technologies. The recombinant active GSK3β (G09-10G) and CK1α (C64-10G) proteins were from SignalChem Lifesciences Corp.
Temozolomide (TMZ), tideglusib, lithium chloride, glutathione S-transferase (GST) beads, cycloheximide (CHX), and MG132 were from Sigma. Puromycin, hygromycin, and D4476 (4-[4-(2,3-dihydro-benzo[1,4]dioxin-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl] benzamide) were from EMD Biosciences. Protein G agarose was from Life Technologies. The recombinant active GSK3β (G09-10G) and CK1α (C64-10G) proteins were from SignalChem Lifesciences Corp.
5
Immunoprecipitation of Myo1g Complexes
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Resting, activated, or capping induced-B cells were lysed with RIPA buffer [20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM PMSF] 30 min at 4°C. After that, the lysates were centrifuged 30 min at 4°C at 18,000 g and the supernatants were mixed with α-Myo1g or NIM-R8, using rabbit IgG or rat IgG as isotype controls, respectively. The supernatants were incubated 4 h at 4°C in agitation; then, the complexes were precipitated with protein G-agarose (Life Technologies), maintaining the temperature at 4°C. Complexes were washed three times with RIPA buffer and boiled in Laemmli buffer. Western blotting was performed under standard conditions.
6
Cell Adhesion and Fibronectin Polymerization
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PureCol bovine type 1 collagen was purchased from Advanced Biomatrix (San Diego, CA). All cell culture components were purchased from Hyclone Laboratories. Protein G agarose and rhodamine–phalloidin were purchased from Life Technologies. Alexa Fluor 488–conjugated fibronectin was purchased from Cytoskeleton. Fibronectin-free serum was a kind gift from Alissa Weaver. Fibronectin polymerization–blocking pUR4B and the control III-11c peptides were a kind gift from J. Sottile and were used at a final concentration of 250 nM.
7
CDKL5 Protein Expression Analysis
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Primary hippocampal neurons were lysed in 3X Laemmli buffer, and samples were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes and blocked in 5% non-fat milk in TBS-T (20 mM of Tris-HCl pH 7.4, 150 mM of NaCl, 0.2% Tween-20). Blots were incubated with primary antibodies overnight at 4 °C, washed in TBS-T and incubated with appropriate secondary antibodies for 1 h at room temperature. Blots were developed with protein detection system-ECL (Genespin) coupled to G:BOX Chemi Imaging System (Syngene). Densitometric expression analyses were performed using ImageJ software.
CDKL5 was immunoprecipitated from 400 μg of HEK293T cells or 1 mg of a mouse brain extract (PND20-30) lysed in lysis buffer [mM: 50 Tris-HCl pH 7.4, 150 NaCl, 1 EDTA, 1 EGTA, 1% Triton X-100, 1X protease inhibitor cocktail (PIC, Sigma-Aldrich, Sant Louis, MO 63103, USA) and 1X PhosSTOP (Roche)] and incubated overnight at 4 °C with 1 µg of anti-CDKL5 or unrelated IgGs as control. The immunocomplexes were precipitated with protein-G agarose (Life Technologies), washed several times with lysis buffer and analysed by SDS-PAGE and western blotting.
CDKL5 was immunoprecipitated from 400 μg of HEK293T cells or 1 mg of a mouse brain extract (PND20-30) lysed in lysis buffer [mM: 50 Tris-HCl pH 7.4, 150 NaCl, 1 EDTA, 1 EGTA, 1% Triton X-100, 1X protease inhibitor cocktail (PIC, Sigma-Aldrich, Sant Louis, MO 63103, USA) and 1X PhosSTOP (Roche)] and incubated overnight at 4 °C with 1 µg of anti-CDKL5 or unrelated IgGs as control. The immunocomplexes were precipitated with protein-G agarose (Life Technologies), washed several times with lysis buffer and analysed by SDS-PAGE and western blotting.
8
Flow Cytometry and Protein Analysis
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Flow cytometry was performed on a FACS Canto II (BD Biosciences) and analyzed using FlowJo Software (Treestar). Cells were stained with antibodies to CD3, CD4 (OKT4), CXCR4 (12G5), VLA-4 (9F10) and CD15s (CSLEX1) from BD biosciences, and HECA452, CD45 (2D1), CD44 (BJ14), CD162 (KPL-1), CD43 (CD43-10G7), and FoxP3 (206D) from Biolegend. Staining with mouse E-selectin Ig chimera (RnD Systems) was detected with either anti-His FITC (Bethyl Laboratories) for flow cytometry or secondary rat anti-mouse CD62E (BBIG-E4, RnD Systems) followed by goat anti-rat IgG HRP (Southern Biotech) for western analyses.
Lysates were made from cells by sonication/vortexing in buffer containing 50 mM Tris, 150 mM NaCl, 20 ug/ml phenylmethanesulfonyl fluoride (PMSF), 0.2% NaN3, protease inhibitor cocktail (Roche), 2% NP40, and 0.2% SDS. Precleared lysates were incubated antibodies to CD43 (1G10, L60, BD Biosciences; 20819, Santa Cruz Biotechnology), PSGL-1 (KPL-1, BD Biosciences; 20929, Santa Cruz Biotechnology), or CD44 (515, BD Biosciences; 2C5, RnD systems). Protein was immunoprecipitated with Protein-G agarose (Life Technologies). Western Blots were run with Reducing SDS-PAGE gels (Bio-Rad).
Lysates were made from cells by sonication/vortexing in buffer containing 50 mM Tris, 150 mM NaCl, 20 ug/ml phenylmethanesulfonyl fluoride (PMSF), 0.2% NaN3, protease inhibitor cocktail (Roche), 2% NP40, and 0.2% SDS. Precleared lysates were incubated antibodies to CD43 (1G10, L60, BD Biosciences; 20819, Santa Cruz Biotechnology), PSGL-1 (KPL-1, BD Biosciences; 20929, Santa Cruz Biotechnology), or CD44 (515, BD Biosciences; 2C5, RnD systems). Protein was immunoprecipitated with Protein-G agarose (Life Technologies). Western Blots were run with Reducing SDS-PAGE gels (Bio-Rad).
9
Immunoprecipitation and Western Blot Analysis
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RIPA buffer (150 mM NaCl, 50 mM Tris-HCl [pH 8.0], 0.5% Nonidet P-40, 5 mM EDTA and a protease and phosphatase inhibitor cocktail (Bimake, Houston, TX, USA) were used to lyse cells. SDS-PAGE resolved the clarified lysates. Samples were transferred to PVDF membranes for Western blots and detected by ECL detection reagents (Beyotime, Shanghai, China). For immunoprecipitation, supernatants were first incubated with anti-FLAG agarose overnight at 4°C. Subsequently, pellets were washed three times with NETN buffer. To examine endogenous interactions, cell lysates were divided into two parts, incubated with either anti-IgG, anti-BCL-2 or anti-PARK2 for 2 h, and then incubated with protein G agarose (Life Technologies, Waltham, MA, USA) overnight. Beads were then washed three times with NETN buffer; and the samples were resolved on SDS-PAGE.
10
Purification and Analysis of Proteins
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