Protein concentration was determined by BCA protein assay for all biochemistry. Neurons were lysed in denaturing buffer for immunoprecipitation: 50 mM Tris HCl, 1 mM EDTA, 1% SDS, 2 mM Na3VO4, 10 mM NaF, 50 mM N-ethylmaleimide, protease inhibitor cocktail (Sigma). Lysates were sonicated and heated at 50°C for 20 min, then diluted 1:5 in RIPA buffer (50 mM Tris HCl pH 7.6, 150 mM NaCl, 1% Igepal, 0.5% Sodium deoxycholate, 1 mM EDTA, 2 mM Na3VO4, 10 mM NaF, 50 mM N-ethylmaleimide, protease inhibitor cocktail). Standard immunoprecipitation procedures were performed using overnight incubation with γ2 subunit antibody or rabbit IgG (sci2027; Sigma), 1 h incubation with Protein A Sepharose 4B beads (Invitrogen), three RIPA buffer washes, and loading for SDS-PAGE. After electrophoresis and transfer to nitrocellulose membrane, samples were probed with primary antibody overnight followed by the appropriate horseradish peroxide (HRP)-coupled secondary antibody.
Protein a sepharose 4b beads
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Protein A-Sepharose 4B beads are a chromatography resin used for the purification of immunoglobulins and other proteins that bind to Protein A. The beads are composed of Sepharose 4B, a cross-linked agarose matrix, with covalently attached Protein A, a bacterial protein that binds to the Fc region of many antibodies. These beads provide a high-capacity and selective medium for the capture and purification of target proteins from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Cellline cl 1000 flasks, by Merck Group (2 mentions)
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
Ha ubiquitin plasmid, by Addgene (2 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Sodium borate buffer ph 9, by Merck Group (1 mentions)
Sodium azide, by Merck Group (1 mentions)
Ethanolamine, by Merck Group (1 mentions)
Polypropylene column, by Bio-Rad (1 mentions)
Anti ubiquitin antibody 3933, by Cell Signaling Technology (1 mentions)
Dimethyl pimelimidate dihydrochloride, by Merck Group (1 mentions)
P gsk3β ser9, by Cell Signaling Technology (1 mentions)
P erk thr202 tyr204, by Cell Signaling Technology (1 mentions)
E cad, by Cell Signaling Technology (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
N cad, by Abcam (1 mentions)
Gsk3β y174, by Abcam (1 mentions)
Horseradish peroxidase hrp labelled secondary antibody, by Beyotime (1 mentions)
Zeb1 d80d3, by Cell Signaling Technology (1 mentions)
13 protocols using protein a sepharose 4b beads
1
Protein Concentration and Immunoprecipitation Assay
2
Isolation of GABAA Receptor Complexes
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Mice were intraperitoneally (I.P.) injected with vehicle control or 10 mg/kg DZP and sacrificed 12 h post-injection (n = 4 mice per treatment). Mouse cortical tissue was homogenized in co-IP buffer (50 mM Tris HCl pH 7.6, 50 mM NaCl, 0.25% Igepal, 1 mM EDTA, 2 mM Na3VO4, 10 mM NaF, 50 mM N-ethylmaleimide, and Sigma protease inhibitor cocktail) using a Dounce homogenizer. Tissue was solubilized with end-over-end mixing at 4°C for 15 min, and then spun at 1,000 g to remove non-solubilized fractions. Each immunoprecipitation tube contained 375 μg of tissue lysate brought up to 1 ml volume using co-IP buffer. Lysates were precleared using Protein A Sepharose 4B beads (Invitrogen) for 1 h at 4°C. Lysate was then immunoprecipitated overnight with 2.5 μg rabbit γ2 subunit antibody (224003, Synaptic Systems) or 2.5 μg rabbit IgG (2027, Santa Cruz). The next day, 40 μl Protein A Sepharose slurry was added and mixed for 2 h at 4°C on a nutator. Beads were then washed 3× at 4°C on a nutator in 1 ml co-IP buffer. Beads were denatured with SDS-PAGE loading buffer [Laemmli Sample buffer (Bio-Rad) + β-mercaptoethanol] with heat at 70°C for 10 min and intermittent vortexing. Two immunoprecipitation reactions were performed per animal and were pooled into a single tube without beads to be used for downstream in-gel digestion.
3
Purification and Crosslinking of W6/32 Antibodies
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W6/32 monoclonal antibodies were purified using protein-A Sepharose 4B beads (Invitrogen, Carlsbad, CA) from the supernatant of HB95 (ATCC® HB-95™) cells grown in CELLLine CL-1000 flasks (Sigma-Aldrich, St. Louis, MI). The antibody quantity was determined on a NanoDrop One spectrophotometer (Thermo Fisher Scientific). For the conventional column-based IP, 5 mg of purified antibodies were mixed with 1 ml of protein-A beads for 1 hour at room temperature, followed by the addition of 20 mM dimethyl pimelimidate (DMP, Sigma-Aldrich) in 0.2 M sodium borate buffer (pH 9, Sigma-Aldrich) for 30 min. The reaction was finally quenched by 0.2 M ethanolamine (pH 8, Sigma-Aldrich), and the crosslinked beads were kept in 0.02% sodium azide (Sigma-Aldrich) at 4°C until use.
4
Immobilization of Pan-specific MHC Class I Antibody
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Pan-specific MHC Class I antibody (W6/32 clone) was ordered from BioXCell. 5 mg of antibody was loaded on 1 mL of Protein A-Sepharose 4B beads (Invitrogen) packed in a polypropylene column (BioRad) and incubated for 30 min. Antibody-bound beads was washed with borate buffer (pH 9) and incubated with 20 mM dimethyl pimelimidate (DMP) linker for 45 min. Crosslinking reaction was stopped by incubating the column with ethanolamine for 2 h. Then the column was washed with phosphate buffer saline and stored in PBS with 0.02% sodium azide at 4 °C. On the day of experiment, column was washed with 0.1 N acetic acid and equilibrated with 100 mM Tris–HCl, pH 8.0.
5
Enrichment of Ubiquitinated Exosomal Proteins
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Ubiquitinated
proteins were enriched using Protein A-Sepharose 4B beads (Invitrogen,
Carlsbad, CA) that had been incubated with anti-ubiquitin antibody
3933 (Cell Signaling Technology, Danvers, MA) in a 1:600 dilution
with rotation for 4 h at 4 °C. Excess antibody was removed from
the beads by washing with 0.8 M urea in 50 mM ammonium bicarbonate
and centrifuging three times at 3000g for 2 min.
One-hundred micrograms of exosome lysate was added to the Sepharose
bead slurry and incubated with rotation overnight at 4 °C. The
unbound fraction was collected via centrifugation at 500g for 5 min. The Sepharose bead slurry was washed with 50 mM ammonium
bicarbonate and centrifuged at 1000g for 5 min to
remove nonspecifically bound proteins. Bound proteins were eluted
by incubating the Sepharose bead slurry in 0.2 M glycine, pH 2.6,
for 1 h at 4 °C and collected via centrifugation at 13 000g for 5 min. The elution was repeated, and the two elution
fractions were combined.22 Enriched fractions
of ubiquitinated exosomal proteins were subsequently processed either
by tryptic digestion in gel or in solution and immunoprecipitation
of peptides with glycinylglycine-modified lysine residues.
proteins were enriched using Protein A-Sepharose 4B beads (Invitrogen,
Carlsbad, CA) that had been incubated with anti-ubiquitin antibody
3933 (Cell Signaling Technology, Danvers, MA) in a 1:600 dilution
with rotation for 4 h at 4 °C. Excess antibody was removed from
the beads by washing with 0.8 M urea in 50 mM ammonium bicarbonate
and centrifuging three times at 3000g for 2 min.
One-hundred micrograms of exosome lysate was added to the Sepharose
bead slurry and incubated with rotation overnight at 4 °C. The
unbound fraction was collected via centrifugation at 500g for 5 min. The Sepharose bead slurry was washed with 50 mM ammonium
bicarbonate and centrifuged at 1000g for 5 min to
remove nonspecifically bound proteins. Bound proteins were eluted
by incubating the Sepharose bead slurry in 0.2 M glycine, pH 2.6,
for 1 h at 4 °C and collected via centrifugation at 13 000g for 5 min. The elution was repeated, and the two elution
fractions were combined.22 Enriched fractions
of ubiquitinated exosomal proteins were subsequently processed either
by tryptic digestion in gel or in solution and immunoprecipitation
of peptides with glycinylglycine-modified lysine residues.
6
Purification and Cross-Linking of Anti-MHC I Antibody
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Anti–MHC I monoclonal antibody was purified from the supernatant of HIB 34.1.2 (gift from Angel Miguel Garcia-Lora, Hospital Universitario Virgen de las Nieves, Granada, Spain) CELLLine CL-1000 flasks (Sigma-Aldrich) using Protein A–Sepharose 4B beads (Invitrogen). Antibodies were cross-linked to Protein A–Sepharose 4B beads at a concentration of 5 mg of antibodies per 1 mL volume of beads. For this purpose, the antibodies were incubated with the Protein A–Sepharose 4B beads for 1 hour at room temperature. Chemical cross-linking was performed by the addition of dimethyl pimelimidate dihydrochloride (Sigma-Aldrich) in 0.2 M sodium borate buffer pH 9 (Sigma-Aldrich) at a final concentration of 20 mM for 30 minutes. The reaction was quenched by incubation with 0.2 M ethanolamine pH 8 (Sigma-Aldrich) for 2 hours. Cross-linked antibodies were kept at 4°C until use.
7
Immunoaffinity Purification of FICD Protein
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The absence of FICD protein in AR42j FICD-/- cells was confirmed by immunoaffinity purification as the endogenous protein could not be detected by immunoblotting of total lysates. Protein extracts from wildtype and FICD-/- AR42j cells were prepared in HG lysis buffer containing protease inhibitors, cleared, normalized and equal volumes of the lysates (4 mg total protein) were incubated with 15 µl UltraLink Hydrazine Resin (Pierce cat. # 53149, UK) on which FICD-specific chicken IgY antibodies have been covalently immobilized according to the manufacturer’s instructions (chicken anti-FICD) or rabbit polyclonal IgG antibodies against human FICD (rabbit anti-FICD; LifeSpan BioSciences) bound to Protein A Sepharose 4B beads (Invitrogen cat. #10-1041), for 16 hr at 4°C. The beads were then recovered by centrifugation for 1 min at 1,000 g and washed three times with HG lysis buffer. Bound proteins were eluted in 40 µl 2 x SDS sample buffer for 10 min at 70°C and equal volumes of the samples were loaded on a 12% SDS polyacrylamide gel, and endogenous FICD was detected by immunoblotting with rabbit anti-FICD or chicken anti-FICD antibodies. Samples of the normalized lysates (25 µg) were loaded and BiP was detected as an “input” control.
8
Cell Signaling Pathway Antibodies
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Antibodies that recognize pAKT (Ser473) (Cell Signaling Technology, #9271), AKT (Cell Signaling Technology, #9272), pERK (Thr202/Tyr204) (Cell Signaling Technology, #4370), ERK (Cell Signaling Technology, #4695), p85α (Santa Cruz, sc-376112), p110α (Cell Signaling Technology, #4249), β-actin (Sigma, A1978), NCAD (Abcam, AB98952), ECAD (Cell Signaling Technology, #3195), VIM (RV202) (Abcam, AB8978), ZEB1 (D80D3) (Cell Signaling Technology, #3396), pGSK3β (Ser9) (Cell Signaling Technology, #9323), GSK3β (Y174) (Abcam, AB32391), and non-phospho CTNNB1 (Ser33/37/Thr41) (Cell Signaling Technology, #4270) were used in the study. Horseradish peroxidase (HRP) labelled secondary antibody (Beyotime Biotech) was used in western blots. Protein A-Sepharose 4B beads (Invitrogen, 10-1041) was used for immunoprecipitation studies.
9
Western Blot and Immunoprecipitation Analysis
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Cells were lysed with radioimmunoprecipitation assay buffer (Beyotime Biotech). Protein was determined, separated and transferred to polyvinylidene difluoride membranes (Pall Life Sciences). The membranes were blocked, then probed with corresponding primary antibody and HRP-conjugated secondary antibodies (Beyotime Biotech), and detected using BeyoECL PLUS Kit (Beyotime Biotech).
For immunoprecipitation assays, cell lysates were incubated with the PIK3R1 antibody overnight and precipitated with protein A-Sepharose 4B beads (Invitrogen) at 4°C for 4 h. The immunoprecipitated proteins were washed, separated on SDS-PAGE, transferred onto a PVDF membrane, and followed by western blot analysis.
For immunoprecipitation assays, cell lysates were incubated with the PIK3R1 antibody overnight and precipitated with protein A-Sepharose 4B beads (Invitrogen) at 4°C for 4 h. The immunoprecipitated proteins were washed, separated on SDS-PAGE, transferred onto a PVDF membrane, and followed by western blot analysis.
10
Isolation and Identification of eIF3f Interactors
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After native electrophoresis, the 120 kDa region of the gel was excised and eluted overnight at 4 °C in 1 mL of ProteoJet Mammalian Cell Lysis Reagent. For non-specific bound protein removal, the elution was incubated 1 h at 4 °C with a non-specific IgG and 100 μL of 50 % Protein A-Sepharose 4B beads (Invitrogen). After centrifugation, the supernatant was incubated at 4 °C with the antibody against eIF3f and 100 μL of 50 % Protein A-Sepharose 4B beads, washed 4 times with lysis buffer, resuspended in sample buffer (2 % SDS, 20 % glycerol, and 0.5 % bromophenol blue in 62 mM Tris HCl buffer, pH 6.8), boiled for 5 min, and subjected to electrophoresis on a 10 % SDS-polyacrylamide gel (10 % SDS-PAGE). A PageRuler Plus Prestained Protein Ladder (Thermo Scientific, Rockford, IL, USA) was included in all SDS-PAGEs. The resolved proteins were detected by Coomassie blue R250 staining (Bio-Rad); the unknown protein bands were excised and sent for N-terminal protein/peptide sequencing (Iowa State University of Science and Technology, Ames, IA, USA). After sequencing service, the partial amino acid sequences were subjected to a NCBI Blastp search to identify possible protein partners of eIF3f.
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