To immunoprecipitate Arc from the synaptoneurosomal fraction, we used PureProteomeTM Protein A/G Mix Magnetic Beads (Millipore, Cat # LSKMAGS08) following the manufacturer’s Protocol B instructions. Briefly, 1 μg/μl of Arc and IgG antibodies (Table 1 ) were incubated with magnetic beads in PBS 1X -0.01% Tween 20 for 45 min at RT with 1000 rpm agitation. Then, the Antibody-Bead complex was washed three times in PBS 1X -0,01% Tween 20 and then incubated with the synaptoneurosomal fraction total lysate, previously quantified, for 4 hours at −20°C in constant agitation. Next, the samples were washed three times with mild lysis buffer (M-RIPA: 50 mM Tris-HCl; 150 mM NaCl; 1% NP-40; 0.25% sodium deoxycholate, 1 mM EDTA), and two times with PBS 1X -0.1% Tween 20. Finally, we added sample buffer and incubated the samples for 10 min at 70°C, rescued the supernatant and then Arc precipitates were analyzed by Western Blot.
Pureproteome protein a g mix magnetic beads
Manufactured by Merck Group
Sourced in United States
The PureProteome™ Protein A/G Mix Magnetic Beads are a type of lab equipment used for the purification and isolation of antibodies and other proteins from biological samples. The beads are coated with a mixture of Protein A and Protein G, which have a high affinity for the Fc region of immunoglobulins. The magnetic properties of the beads allow for easy separation and washing of the captured proteins using a magnetic field.
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Lab products found in correlation
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Ecl reagent, by GE Healthcare (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Acetylated lysine, by Cell Signaling Technology (2 mentions)
Capture socs3 antibody, by Abcam (1 mentions)
Rabbit anti socs3 antibody, by Abcam (1 mentions)
Sds page sample loading buffer, by Beyotime (1 mentions)
Anti polii antibody, by Merck Group (1 mentions)
Ecl western blot reagent, by Thermo Fisher Scientific (1 mentions)
Mouse anti carm1 antibodies, by Cell Signaling Technology (1 mentions)
Supersignal west femto substrate, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Saf32, by Cayman Chemical (1 mentions)
Anti myc, by Merck Group (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Anti occludin antibody, by Thermo Fisher Scientific (1 mentions)
36 protocols using pureproteome protein a g mix magnetic beads
1
Immunoprecipitation of Arc from Synaptoneurosomal Fraction
2
SOCS3 Protein Immunoprecipitation and Detection
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Immunoprecipitation was conducted using PureProteomeTM Protein A/G Mix Magnetic Beads (Millipore). Briefly, the macrophages were lysed using lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1% Triton, 1 mM EGTA, 1 mM NA3VO4) supplemented with 1% protease/phosphataes inhibitor cocktail (CST), and incubated on ice for 5 minutes. The supernatant was collected by centrifugation at 14,000 g for 10 minutes, and co-incubated with capture SOCS3 antibody (5 μg/107 cells; Abcam) at 4°C overnight. The antibody-antigen complex was then added to the beads and incubated for 30 min at room temperature. Finally, adding the appropriate elution buffer for denaturing elution after washing the complex. The samples were heated to 95–100°C for 5 minutes, and then cooled on ice. Immunoprecipitated proteins were analyzed by western blot with a rabbit-anti-SOCS3 antibody (Abcam). To eliminate the interference of rabbit lgG heavy and light chains, Mouse Anti-rabbit lgG (Conformation Specific) (CST), which only reacts with native lgG and doesn’t bind to the denatured and reduced rabbit lgG heavy and light chains, were used as the secondary antibody for western blot.
3
Quantifying Pol II-pCDK9 Interactions
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HELF or HEK293 cells growing in 100 mm plates were infected with different HCMV strains at an MOI of 1.0 or transfected with vectors transcribing RNA2.7C2c. PureProteomeTMProteinA/G Mix Magnetic Beads (Millipore, #LSKMAGAG10) were coated with anti-Pol II antibody (Millipore, #05-623-Z) and were incubated with lysates at 4 °C overnight. The captured protein complex was eluted with 60 μL SDS-PAGE sample loading buffer (Beyotime, Nantong, China, #P0015) and was then heated at 70 °C for 10 min. After the beads were removed, the supernatants were loaded on 8% SDS-PAGE gel. Blotted PVDF membranes were incubated with antibodies against Pol II and pCDK9, followed by peroxidase-conjugated goat anti-mouse or rabbit IgG (ZSGB-BIO, Guangzhou, China, #ZB-2305 or #ZB-2301) with ECL Western blot reagent (ThermoFisher). Protein relative densities were quantified using ImageJ software 1.44p (NIH). Pol II captured was measured as a quantitative reference to calculate the relative amount of pCDK9 binding to Pol II.
4
Immunoprecipitation of Protein Complexes
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Cells were washed three times with ice-cold PBS and resuspended in lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and Complete Protease Inhibitor Mixture). 250 μg of cell extract were incubated in a rotating mixer for 2 h with the primary antibody at 4 °C, followed by an overnight incubation with PureProteomeTM Protein A/G mix magnetic beads (Millipore). Samples were washed as indicated by the manufacturer, and immunoprecipitated proteins were eluted with freshly prepared IP buffer (10% glycerol, 50 mm Tris-HCl, pH 6.8, and 1 m NaCl) by incubating at room temperature for 1 h on a rotating mixer. Samples were analyzed by SDS-PAGE and Western blotting.
5
Immunoprecipitation of CARM1 from Embryonic Brains
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Embryonic brains were collected at E16.5 and homogenized in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF, 1X protease, and phosphatase inhibitor cocktail). The samples were centrifuged to collect the supernatant. BCA protein assay was performed to measure protein concentration in the supernatant. Approximately 2 mg of protein was used for immunoprecipitation using 1 µl of mouse anti-CARM1 antibodies (Cell Signaling Technologies, #12495; RRID:AB_2797935), which were crosslinked to PureProteome Protein A/G mix magnetic beads (Millipore). The eluted samples were separated on SDS-PAGE gel and transferred onto a PVDF membrane. The membrane was incubated with blocking solution (5% milk, 1X TBS, 0.1% Tween-20) for 2 h, with mouse anti-CARM1 antibodies (1:2,000) for overnight at 4°C, then with HRP-conjugated antimouse secondary antibodies for 2 h at room temperature. SuperSignal West Femto substrate (Thermo Fisher Scientific) was used for detection. The lysates were also analyzed by western blotting using anti-GAPDH (Cell Signaling Technologies, #5174, RRID:AB_10622025; 1:2,000) to show the same amount of protein was used for IP.
6
Immunoprecipitation of PrP^C and Myc-mGluR5
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The co‐IP of PrPC and Myc‐mGluR5 was previously described.12 HEK‐293T cells were maintained in Dulbecco's Modified Eagle Medium (DMEM), supplied with 10% fetal calf serum (FCS), 1% L‐glutamine (2 mmol/L), 1% sodium pyruvate (1 mmol/L), and 1% penicillin/streptomycin (100 U/mL). Cells were transfected for 36 h using Lipofectamine 2000 transfection reagent (Invitrogen). AZ59, 6D11, and control IgG were applied to the cells for 3 h before 1 μmol/L Aβo was added to the plates for an additional hour; all treatment was done at 4˚C. To prepare detergent solubilized cell lysates, cells were rinsed with ice‐cold PBS and solubilized in RIPA buffer containing complete protease inhibitor cocktail (Roche) and phosSTOP phosphatase inhibitor cocktail (Roche). The insoluble fraction was removed by centrifugation at 20,000g and the supernatant was used for immunoprecipitation. One microgram of capture antibody was incubated overnight at 4°C with 1 mg of detergent solubilized lysate protein with continuous mixing. The antibodies used were anti‐Myc (SigmaAldrich, C3956) and SAF32 (Cayman, 189720) with capture by PureProteome Protein A/G Mix Magnetic Beads (Millipore, LSKMAGAG10). After incubation, beads were washed three times in wash buffer prior to elution of proteins in SDS‐PAGE sample loading buffer.
7
C. elegans Protein Extraction and Analysis
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To prepare C. elegans proteins, synchronized young adult worms were grown on a 9.5 cm plates at 20 °C, and washed off from the plates with M9 buffer. The worms were lysed by sonication in lysis buffer (50mMTris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, phosphatase inhibitors, and protease inhibitors) and then immunoprecipitated with anti-GFP antibody, followed by western blotting. For IP assay, total protein lysates were incubated overnight with corresponding antibodies with gentle shacking at 4 °C., followed by addition of 40 μl of Pure Proteome protein A/G Mix Magnetic Beads (Millipore) for another 3 h. The beads were resuspended in 60 μl of 2 × loading buffer and boiled for 10 min. Then the supernatant was subjected to SDS-PAGE, transferred to polyvinylidene fluoride membrane (Millipore) and visualized by using appropriate primary antibodies coupled with HRP-conjugated secondary antibodies by ECL reagent (GE Healthcare).
8
PCB153 Modulation of Tight Junction Integrity
9
Western Blot and Co-Immunoprecipitation Protocol
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Western blot analysis was performed using whole cell extracts prepared by lysing the cells in lysis buffer (pH 8.0) containing 50 mM Tris–HCl, 150 mM NaCl, 5 mM EDTA, 1% NP40, 0.05% PMSF, 2 mg/mL aprotinin, and 2 mg/mL leupeptin. Antibodies against GAPDH and β-actin were used to assess the purity of the respective fractions.
For the co-immunoprecipitation analyses, the cells were resuspended in Nonidet P40 lysis buffer containing 10 mM nicotinamide and 10 μM TSA. Cell lysates were mixed with antibodies at 4 °C overnight, and PureProteome™ Protein A/G Mix Magnetic Beads (Millipore Corporation) were then added. The detailed protocol for this procedure can be found atwww.millipore.com . The proteins were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred onto polyvinylidene fluoride membranes, which were blocked in 5% (w/v) nonfat milk and hybridized with specific primary antibodies. The resulting protein bands were visualized using ECL (Thermo Scientific) after hybridization with a secondary antibody (Thermo Scientific). All of the western blot results were quantified using Image J. We repeated these experiments three times.
For the co-immunoprecipitation analyses, the cells were resuspended in Nonidet P40 lysis buffer containing 10 mM nicotinamide and 10 μM TSA. Cell lysates were mixed with antibodies at 4 °C overnight, and PureProteome™ Protein A/G Mix Magnetic Beads (Millipore Corporation) were then added. The detailed protocol for this procedure can be found at
10
Co-immunoprecipitation and Western Blot
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For co‐immunoprecipitation (Co‐IP), total cell extracts were prepared in IP lysis buffer (Vazyme, Nanjing, China) supplemented with protease inhibitor (Roche, Indianapolis, IN, USA). Aimed proteins were immunoprecipitated with the indicated target‐specific antibody (1 µg) overnight at 4 °C. Next day, the samples were incubated with 40 µL PureProteome™ Protein A/G Mix Magnetic Beads (Millipore) for 30 min at room temperature and then were washed with PBS containing 0.1% Tween 20 (pH 7.4) for five times before denaturation. Finally, the immunoprecipitated samples were identified by western blot with their corresponding primary antibodies.
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