An in vitro enzyme activity assay for wild type IDH1 was performed as previously described (8 (link)). In brief, 20 ng purified IDH1 and YF variant proteins that were pre-treated with or without recombinant active tyrosine kinases in an in vitro kinase assay were added to 100 μl enzyme activity assay buffer (25mM Tris-HCl (pH7.5), 10 mM MgCl2, 5 mM DTT) containing 0.5 mM NADP+ (Sigma-Aldrich) and 1 mM isocitric acid (Sigma-Aldrich). To determine the wild type IDH1 activity in cells, endogenous IDH1 from 1×107 cells was bound to protein G-Sepharose 4 Fast Flow beads (Sigma-Aldrich) by immunoprecipitation using IDH1 antibody (CST). To test IDH1 activity in xenograft tumor tissues, around 2 mg of total tumor lysates were used. After immunoprecipitation, the IDH1-bound beads were washed 3 times and eluted using IDH1 peptide (CST). Then 10 ul of the supernatant was added to 100μl assay buffer (25mM Tris-HCl, pH7.5, 10 mM MgCl2, 5 mM DTT) containing 0.5 mM NADP+ and 1 mM isocitric acid. IDH1 activity was measured by recording absorbance of 340 nm in kinetic mode every 20 seconds for 15 minutes using a SpectraMax Plus spectrophotometer (Molecular Devices).
Protein g sepharose 4 fast flow beads
Manufactured by Merck Group
Sourced in Germany
Protein G Sepharose 4 Fast Flow beads are a chromatography matrix used for the purification of antibodies and antibody-containing samples. The beads are composed of highly cross-linked agarose and recombinant Protein G, which binds to the Fc region of immunoglobulins. The fast flow property of the beads allows for efficient and rapid purification.
Automatically generated - may contain errors
Lab products found in correlation
Complete protease inhibitor cocktail, by Merck Group (2 mentions)
Spectramax plus spectrophotometer, by Molecular Devices (1 mentions)
Isocitric acid, by Merck Group (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Anti flag m2 affinity gel, by Merck Group (1 mentions)
Ab81321, by Abcam (1 mentions)
Mouse anti e cadherin, by BD (1 mentions)
Tc18 sep pak column, by Waters Corporation (1 mentions)
Sorbent, by Waters Corporation (1 mentions)
7 protocols using protein g sepharose 4 fast flow beads
1
Quantitative IDH1 Enzyme Activity Assay
2
Co-immunoprecipitation of Flag and GFP Fusions
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For coIP of Flag fusions, the respective HEK293 stable lines were incubated with tetracycline (1 µg/ml) for 24 h, after which they were washed with 1x PBS, harvested, and frozen at −80°C or lysed immediately (50 mM Hepes, pH 8, 100 mM KCl, 2 mM EDTA, 10% glycerol, 0.1% NP-40, 1 mM DTT, and protease inhibitors) for 30 min on ice. The lysates were then frozen in dry ice for 5 min and then thawed and centrifuged for 20 min at 16,000 ×g at 4°C. The cleared lysates were then incubated with ANTI-FLAG M2 Affinity Gel (Sigma-Aldrich) overnight at 4°C. A fraction of the protein extracts (inputs) were saved before the incubation with the beads. After incubation, beads were pelleted and washed with lysis buffer. Samples (inputs and IPs) were prepared for SDS-PAGE by addition of Laemmli buffer and boiling. Proteins were transferred to polyvinylidene fluoride membranes (Immobilon-P; Millipore) and probed with antibodies to detect the FLAG fusions and endogenous proteins. The GFP coIPs were performed similarly using extracts from HEK293 or RPE-1 cells expression GFP-fusions. Protein G Sepharose 4 Fast Flow beads (P3296; Sigma-Aldrich) were incubated with 2 µg GFP antibody raised in Goat for 2 h at 4°C and then washed with lysis buffer. GFP antibody-conjugated beads were then used to pull down the GFP fusions. Primary and secondary antibodies used are listed in Table S2 .
3
Immunoprecipitation of NRP1 from Murine Splenocytes
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Ten million of activated Murine splenocytes where washed with PBS at 4°c, then lysed in buffer: EDTA 1mM, NaCl150mM, Tris HCl 25mM, glycerol 10%. Antiproteases, antiphosphatases and detergent (final concentration 0.1% NP40) were added extemporaneously. Lysate was centrifuged at 20,000 G for 15 min at 4°c to remove debris. Preclearing was performed: 15μL of Protein G Sepharose 4 Fast Flow beads (Sigma-Aldrich) were added to the lysate and incubated for 1h at 4°c under rotating wheel. Supernatant was divided in two conditions for incubation with 2μG of antibodies: either rabbit anti-mouse NRP1 (ab81321, AbCam) or rabbit IgG control, for 1h at 4°c under rotating wheel. Then, lysates were respectively incubated with 15μL of Protein G Sepharose for 90 min at 4°c under rotating wheel. Beads were washed with PBS at 4°c. Protein complexes were eluted by adding 4X Laemmli buffer followed by heating at 95°c for 5 min. Beta-mercaptoethanol was added and the whole eluate was reheated for 1min and centrifugated. Proteins were resolved on 10% PAGE and analyzed by immunoblotting. Antibodies used included rabbit anti-mouse NRP1 (ab81321, AbCam) and rabbit Anti mouse PD1 (PRS4065, Sigma-Aldrich), diluted in TTBS BSA 5%.
4
Affinity Purification and Immunoprecipitation of E-cadherin and p62
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MDA231-E-cad-Strep-Tag and MDA231-p62-Strep-Tag cells were lysed with Pierce Lysis Buffer. Lysates were incubated with Strep-Tactin Sepharose 50% suspension (IBA Lifesciences) and, after washing, bound proteins were retrieved with Laemmli buffer and analyzed in western blot.
Immunoprecipitation (IP) of endogenous proteins was performed by lysing cells in PLB buffer (20 mM Tris pH 7.5, 200 mM NaCl, 1 mM EDTA, 0.5% Igepal) supplemented with Complete Protease Inhibitor Cocktail (Sigma-Aldrich) and PMSF. Protein lysates (0.5 mg) were immunoprecipitated overnight with the mouse anti-SQSTM1/p62 antibody (Santa Cruz Biotechnology) and then conjugated to Protein G Sepharose 4 Fast Flow beads (Sigma-Aldrich) for 2 h. Immunocomplexes were washed five times with PLB buffer, resuspended in Laemmli buffer containing β-mercaptoethanol and heated at 100°C for 10 min prior gel loading. Proteins were resolved by SDS–PAGE (4–15% gradient). E-cadherin and SQSTM1/p62 were immunodetected with the mouse anti-E-cadherin (BD Biosciences) and the rabbit anti-SQSTM1/p62 (Thermo Fisher Scientific) antibodies, respectively.
Immunoprecipitation (IP) of endogenous proteins was performed by lysing cells in PLB buffer (20 mM Tris pH 7.5, 200 mM NaCl, 1 mM EDTA, 0.5% Igepal) supplemented with Complete Protease Inhibitor Cocktail (Sigma-Aldrich) and PMSF. Protein lysates (0.5 mg) were immunoprecipitated overnight with the mouse anti-SQSTM1/p62 antibody (Santa Cruz Biotechnology) and then conjugated to Protein G Sepharose 4 Fast Flow beads (Sigma-Aldrich) for 2 h. Immunocomplexes were washed five times with PLB buffer, resuspended in Laemmli buffer containing β-mercaptoethanol and heated at 100°C for 10 min prior gel loading. Proteins were resolved by SDS–PAGE (4–15% gradient). E-cadherin and SQSTM1/p62 were immunodetected with the mouse anti-E-cadherin (BD Biosciences) and the rabbit anti-SQSTM1/p62 (Thermo Fisher Scientific) antibodies, respectively.
5
Immunoprecipitation and Sumo-MEK1 Analysis
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IP analyses were carried out as previously described (17 (link)). The cells were suspended and lysed in 50mM Tris-HCl buffer (pH 7.5) with 0.5% Triton X-100 and protease inhibitors (XinShengyuan). Then, anti-MEK1 antibodies (cat. no. ab32091; Abcam) were added to induce cellular lysis and placed on a rotating stirrer at 4˚C for 2 h. Then, protein-G Sepharose 4 Fast Flow beads (30 µl; Sigma-Aldrich; Merck KGaA) were added according to the manufacturer's protocol. The solutions were rotated overnight at 4˚C, and the beads were washed 3 times with Tris-HCl buffer (50 mM; pH 7.5) with 0.5% Triton X-100. In one experiment, the immune complexes were solubilized in SDS-PAGE loading buffer and separated on SDS-PAGE gels (12%) following western blotting to detect sumo-MEK1 with anti-SUMO antibodies (cat. no. ab219724; Abcam).
6
Hybridoma antibody purification and concentration
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The 139H2 in the hybridoma culture supernatant was a kind gift from John Hilkins from The Netherlands Cancer Institute (NKI). The 139H2 was purified with Protein G Sepharose 4 Fast Flow beads (Merck), washed with PBS, eluted with 0.2 mM glycine buffer, pH 2.5, neutralized with 1 M Tris–HCL, pH 8, and dialyzed against PBS with Pierce Protein Concentrators PES, 30 kD MWCO.
7
Immunopeptidomics Sample Preparation
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One hundred million cells per sample were lysed with a buffer containing 0.25% (w/v) sodium deoxycholate, 0.2 mM iodoacetamide, 1 mM EDTA, 1 × cOmplete Protease Inhibitor Cocktail (Merck, Taufkirchen, Germany), 1 mM Phenylmethyl-sulfonylfluoride and 1% Octyl-beta-D-glucopyranoside in PBS [28 (link)]. Then, 10 µg lysate per sample was used for a parallel proteome analysis.
The lysates for the immunopeptidomics analysis were cleared via centrifugation (51,200× g for 50 min at 4 °C), and native MHC class I complexes were precipitated with the pan-MHC class I-specific antibody W6/32 (HB-95, ATCC/ LGC Standards, Wesel, Germany) coupled to protein G sepharose 4 Fast Flow beads (Merck, Taufkirchen, Germany) as described [28 (link)]. Co-precipitated immunopeptides were eluted with 1% (v/v) trifluoroacetic acid (TFA) and purified using Sep-Pak tC18 columns containing 100 mg sorbent (Waters, Eschborn, Germany). The elution of peptides from the tC18 sorbent was conducted in two steps: first with 28% acetonitrile (ACN) in 0.1% TFA and consecutively with 32% ACN in 0.1% TFA. Both eluates were volume-reduced using a vacuum evaporator until almost all liquid was evaporated. The peptides were then resolved with 2% ACN in 0.5% TFA and stored at −80 °C until further analysis [28 (link)].
The lysates for the immunopeptidomics analysis were cleared via centrifugation (51,200× g for 50 min at 4 °C), and native MHC class I complexes were precipitated with the pan-MHC class I-specific antibody W6/32 (HB-95, ATCC/ LGC Standards, Wesel, Germany) coupled to protein G sepharose 4 Fast Flow beads (Merck, Taufkirchen, Germany) as described [28 (link)]. Co-precipitated immunopeptides were eluted with 1% (v/v) trifluoroacetic acid (TFA) and purified using Sep-Pak tC18 columns containing 100 mg sorbent (Waters, Eschborn, Germany). The elution of peptides from the tC18 sorbent was conducted in two steps: first with 28% acetonitrile (ACN) in 0.1% TFA and consecutively with 32% ACN in 0.1% TFA. Both eluates were volume-reduced using a vacuum evaporator until almost all liquid was evaporated. The peptides were then resolved with 2% ACN in 0.5% TFA and stored at −80 °C until further analysis [28 (link)].
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