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Millipore buffer a
Buffer A, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 99 stars, based on 30 article reviews
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buffer a - by Bioz Stars, 2020-04
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Related Articles

Transduction:

Article Title: A Novel Role of Lactosylceramide in the Regulation of Lipopolysaccharide/Interferon-γ-Mediated Inducible Nitric Oxide Synthase Gene Expression: Implications for Neuroinflammatory Diseases
Article Snippet: After incubation with antiserum against iNOS (BD Biosciences PharMingen, San Diego, CA) or H-Ras (Upstate Biotechnology, Lake Placid, NY) or phospho-specific ERK1/2 or pIκB (Signal Transduction) in PVDF buffer for 2 hr at room temperature, the filters were washed three times with TBST buffer and then incubated with horseradish peroxidase-conjugated anti-rabbit or mouse IgG for 1 hr. .. Cells were harvested, washed twice with ice-cold TBS, and lysed in 400 μl of buffer A containing (in m m ): 10 KCl, 2 MgCl2 , 0.5 dithiothreitol plus protease inhibitor mixture (Sigma) and 0.1% Nonidet P-40 in 10 m m HEPES, pH 7.9, for 10 min on ice.

Centrifugation:

Article Title: A Novel Role of Lactosylceramide in the Regulation of Lipopolysaccharide/Interferon-γ-Mediated Inducible Nitric Oxide Synthase Gene Expression: Implications for Neuroinflammatory Diseases
Article Snippet: Cells were harvested, washed twice with ice-cold TBS, and lysed in 400 μl of buffer A containing (in m m ): 10 KCl, 2 MgCl2 , 0.5 dithiothreitol plus protease inhibitor mixture (Sigma) and 0.1% Nonidet P-40 in 10 m m HEPES, pH 7.9, for 10 min on ice. .. After centrifugation at 5000 × g for 10 min the pelleted nuclei were washed with buffer A without Nonidet P-40 and resuspended in 40 μl of buffer B containing 25% (v/v) glycerol, 0.42 m NaCl, and (in m m ) 1.5 MgCl2 , 0.2 EDTA, 0.5 dithiothreitol, and Complete protease inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, IN) in 20 m m HEPES, pH 7.9, for 30 min on ice.

Filtration:

Article Title: Transmembrane pores formed by syntheticp-octiphenyl ?-barrels with internal carboxylate clusters: Regulation of ion transport by pH and Mg2+- complexed 8-aminonaphthalene-1,3,6-trisulfonate
Article Snippet: .. A solution of EYPC (50 mg in 50 μl of EtOH; Avanti Polar Lipids) and sodium cholate (22.4 mg; Sigma) in 950 μl of buffer A was dialyzed against 50 ml of buffer A [ > 6 h, 5 mM N -[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES) (Sigma)/12.5 mM 8-aminonaphtalene-1,3,6-trisulfonate (ANTS)/45.0 mM p -xylenebis(pyridinium)bromide (DPX) (Molecular Probes)/20 mM KCl, pH 7.0 ( )] and 1,000 ml of buffer B [ > 12 h, 5 mM TES/100 mM KCl, pH 7.0 ( )], purified by gel filtration (Sephadex G-50, buffer B), and diluted to 6 ml. .. ANTS/DPX-efflux was recorded as a function of time by measuring changes in emission intensity It [λem = 510 nm, λex = 353 nm ( , ), FluoroMax-2; Jobin-Yvon-Spex, Longjumeau, France] after addition of these EYPC-SUVs (10 mM EYPC, 50 μl) to stirred thermostated buffer C (1.95 ml/10 mM Mes/100 mM KCl, pH 4.5–7.0) to give emission intensity I 0 .

Autoradiography:

Article Title: A Novel Role of Lactosylceramide in the Regulation of Lipopolysaccharide/Interferon-γ-Mediated Inducible Nitric Oxide Synthase Gene Expression: Implications for Neuroinflammatory Diseases
Article Snippet: The membranes were detected by autoradiography, using ECL-plus (Amersham Biosciences, Arlington Heights, IL) after being washed with TBST buffer. .. Cells were harvested, washed twice with ice-cold TBS, and lysed in 400 μl of buffer A containing (in m m ): 10 KCl, 2 MgCl2 , 0.5 dithiothreitol plus protease inhibitor mixture (Sigma) and 0.1% Nonidet P-40 in 10 m m HEPES, pH 7.9, for 10 min on ice.

Immunostaining:

Article Title: Phosphatidylinositol 3-Phosphate 5-Kinase, FAB1/PIKfyve Kinase Mediates Endosome Maturation to Establish Endosome-Cortical Microtubule Interaction in Arabidopsis 1Phosphatidylinositol 3-Phosphate 5-Kinase, FAB1/PIKfyve Kinase Mediates Endosome Maturation to Establish Endosome-Cortical Microtubule Interaction in Arabidopsis 1 [OPEN]
Article Snippet: Paragraph title: Immunostaining of PIN2 ... In brief, 4-d-old seedlings were fixed in 4% paraformaldehyde and 0.25% glutaraldehyde in buffer A (20 m m PIPES [pH 6.8], 137 m m NaCl, and 2.7 m m KCl) under vacuum conditions for 45 min, and then cells were permeabilized by incubation with 2% driselase at room temperature for 30 min and a solution of buffer A containing 50 m m NH4 Cl for 40 min. After a preincubation with buffer A containing 3% bovine serum albumin , the samples were incubated for 16 h in the primary antibodies diluted in buffer A containing 3% at 37°C, and after washing the samples in buffer A, they were incubated for 3 h in Alexa 555-coupled secondary antibodies (Sigma-Aldrich) diluted 1:500 in buffer A and 3% .

Electrophoresis:

Article Title: A Novel Role of Lactosylceramide in the Regulation of Lipopolysaccharide/Interferon-γ-Mediated Inducible Nitric Oxide Synthase Gene Expression: Implications for Neuroinflammatory Diseases
Article Snippet: Total cellular protein (50 μg) was resolved by electrophoresis on 8 or 12% polyacrylamide gels, electro-transferred to a polyvinylidene difluoride (PVDF) filter, and blocked with Tween 20 containing Tris-buffered saline (TBST; 10 m m Trizma base, pH 7.4, 1% Tween 20, and 150 m m NaCl) with 5% skim milk. .. Cells were harvested, washed twice with ice-cold TBS, and lysed in 400 μl of buffer A containing (in m m ): 10 KCl, 2 MgCl2 , 0.5 dithiothreitol plus protease inhibitor mixture (Sigma) and 0.1% Nonidet P-40 in 10 m m HEPES, pH 7.9, for 10 min on ice.

Incubation:

Article Title: Loss of the Notch effector RBPJ promotes tumorigenesis
Article Snippet: In brief, the cells were washed two times with PBS, resuspended in four pellet volumes of buffer A (10 mM Hepes-KOH, pH 7.8, 10 mM KCl, 1.5 mM MgCl2 , 5 mM with addition of a protease inhibitor cocktail; all from Sigma-Aldrich), pelleted for 15 s at 12,000 g and resuspended in 375 µl buffer A containing 0.5% NP-40 (Sigma-Aldrich). .. After mixing, cells were incubated at 4°C for 10 min to lyse the cells, centrifuged for 10 min at 12,000 g at 4°C, and the cytosolic extract was removed.

Article Title: Phosphatidylinositol 3-Phosphate 5-Kinase, FAB1/PIKfyve Kinase Mediates Endosome Maturation to Establish Endosome-Cortical Microtubule Interaction in Arabidopsis 1Phosphatidylinositol 3-Phosphate 5-Kinase, FAB1/PIKfyve Kinase Mediates Endosome Maturation to Establish Endosome-Cortical Microtubule Interaction in Arabidopsis 1 [OPEN]
Article Snippet: .. In brief, 4-d-old seedlings were fixed in 4% paraformaldehyde and 0.25% glutaraldehyde in buffer A (20 m m PIPES [pH 6.8], 137 m m NaCl, and 2.7 m m KCl) under vacuum conditions for 45 min, and then cells were permeabilized by incubation with 2% driselase at room temperature for 30 min and a solution of buffer A containing 50 m m NH4 Cl for 40 min. After a preincubation with buffer A containing 3% bovine serum albumin , the samples were incubated for 16 h in the primary antibodies diluted in buffer A containing 3% at 37°C, and after washing the samples in buffer A, they were incubated for 3 h in Alexa 555-coupled secondary antibodies (Sigma-Aldrich) diluted 1:500 in buffer A and 3% . .. Polyphosphoinositide levels were measured according to .

Article Title: A Novel Role of Lactosylceramide in the Regulation of Lipopolysaccharide/Interferon-γ-Mediated Inducible Nitric Oxide Synthase Gene Expression: Implications for Neuroinflammatory Diseases
Article Snippet: After incubation with antiserum against iNOS (BD Biosciences PharMingen, San Diego, CA) or H-Ras (Upstate Biotechnology, Lake Placid, NY) or phospho-specific ERK1/2 or pIκB (Signal Transduction) in PVDF buffer for 2 hr at room temperature, the filters were washed three times with TBST buffer and then incubated with horseradish peroxidase-conjugated anti-rabbit or mouse IgG for 1 hr. .. Cells were harvested, washed twice with ice-cold TBS, and lysed in 400 μl of buffer A containing (in m m ): 10 KCl, 2 MgCl2 , 0.5 dithiothreitol plus protease inhibitor mixture (Sigma) and 0.1% Nonidet P-40 in 10 m m HEPES, pH 7.9, for 10 min on ice.

Expressing:

Article Title: Rad50 ATPase activity is regulated by DNA ends and requires coordination of both active sites
Article Snippet: Insect cells expressing complexes were lysed in Ni-A buffer (50 mM KH2 PO4 , 10% glycerol, 2.5 mM imidazole, 20 mM β-mercaptoethanol, 0.5 M KCl) using homogenization followed by sonication. .. The eluate was diluted with Buffer A (25 mM Tris pH 8.0, 100 mM NaCl, 10% glycerol, 1 mM DTT) to 150 mM NaCl, and applied to 1 ml anti-FLAG M2 antibody resin (Sigma), washed with Buffer A, with five volumes of 0.5 M LiCl and eluted with 0.1 mg/ml FLAG peptide (Sigma) in Buffer A. Aliquots were flash frozen and stored at −70°C.

Article Title: The Cysteine-rich Domain of the DHHC3 Palmitoyltransferase Is Palmitoylated and Contains Tightly Bound Zinc *
Article Snippet: TriEx Sf9 insect cells expressing DHHC3-FLAG-His WT or DHHC3 mutants were disrupted in lysis buffer (50 m m Tris, pH 7.4, 200 m m NaCl, 1% n -dodecyl-β- d -maltoside (DDM), 10% glycerol, 1 m m TCEP, and protease inhibitors). .. Elutions were pooled and rotated with ANTI-FLAG® M2-agarose affinity gel (Sigma) equilibrated with Buffer A (50 m m Tris, pH 7.4, 100 m m NaCl, 10% glycerol, 0.1% DDM, 1 m m EDTA, 5 μg/ml leupeptin, and 1 μ m pepstatin A) at 4 °C for 2 h. The FLAG affinity resin was washed five times with a total of 50 column volumes of Buffer A and eluted with 5 × 1 column volumes of Buffer A + 0.3 mg/ml FLAG peptide (Sigma).

Chromatography:

Article Title: In vitro assays for targeting and insertion of tail-anchored proteins into the ER membrane
Article Snippet: .. E. coli S30 extract containing tRNACUA Opt ( ; ; ) 250 µM RF-1 aptamer ( ) 40–80 µM Tagless T7 RNA polymerase ( ) 500–600 µM Tagless CmRS(D286R) ( ) 150–200 µM His6 -tagged Sgt2 ( ) Small molecule mix (SMM) (see Recipe) 10 mM amino acid mix (−Met or +Met) (see Recipe) 40 mM 7-hydroxycoumaryl ethylglycine (Cm) (Millipore-Sigma, cat# 792551) Plasmid encoding protein of interest (POI), such as a model TA (SUMO-Bos1-opsin) under control of the T7 promoter (pUC19-Strep3 -SUMO-Bos1-opsin or pUC19-Strep3 -SUMO-Amb-Bos1-opsin) ( ) 11 mCi/mL 35 S-Met (Perkin Elmer, cat#NEG009T001MC) Buffer A (see Recipe) Fixing solution (see Recipe) 3M Potassium acetate (KOAc) dissolved in Buffer A (Millipore-Sigma, cat#P5708) 2M Imidazole dissolved in Buffer A (pH~7.0) (Millipore-Sigma, cat#I5513) Maxi-prep kit (QIAGEN, cat#12963) sterile 100×15mm petri dish 0.6 mL microfuge tubes (amber and clear) 15 mL Falcon tubes Aluminum foil Ni-NTA agarose (QIAGEN, cat# 30310) Amicon Ultra-15 Centrifugal Filter, 10 kDa cutoff (Millipore-Sigma, cat# UFC901024) 10 mL Poly-Prep® Chromatography Column (Bio-Rad, cat#7311550) 12.5% Tris-Glycine SDS-PAGE gels 30 °C water bath 30 °C benchtop incubator TLX-Optima ultracentrifuge (Beckman Coulter Inc.) or equivalent TLA100 rotor (Beckman Coulter Inc.) or equivalent Gel dryer Storage phosphor screen (GE Healthcare Life Sciences) Typhoon Phosphorimager (GE Healthcare Life Sciences) Refrigerated benchtop swinging bucket centrifuge (Beckman Coulter Inc.) Note 1: All reagents and apparatus that come into contact with the IVT reaction should be nuclease-free following standard procedures . ..

Article Title: In vitro assays for targeting and insertion of tail-anchored proteins into the ER membrane
Article Snippet: .. E. coli S30 extract containing tRNACUA Opt ( ; ; ) 250 µM RF-1 aptamer ( ) 40–80 µM Tagless T7 RNA polymerase ( ) 500–600 µM Tagless CmRS(D286R) ( ) 150–200 µM His6 -tagged Sgt2 ( ) Small molecule mix (SMM) (see Recipe) 10 mM amino acid mix (−Met or +Met) (see Recipe) 40 mM 7-hydroxycoumaryl ethylglycine (Cm) (Millipore-Sigma, cat# 792551) Plasmid encoding protein of interest (POI), such as a model TA (SUMO-Bos1-opsin) under control of the T7 promoter (pUC19-Strep3 -SUMO-Bos1-opsin or pUC19-Strep3 -SUMO-Amb-Bos1-opsin) ( ) 11 mCi/mL 35 S-Met (Perkin Elmer, cat#NEG009T001MC) Buffer A (see Recipe) Fixing solution (see Recipe) 3M Potassium acetate (KOAc) dissolved in Buffer A (Millipore-Sigma, cat#P5708) 2M Imidazole dissolved in Buffer A (pH~7.0) (Millipore-Sigma, cat#I5513) Maxi-prep kit (QIAGEN, cat#12963) sterile 100×15mm petri dish 0.6 mL microfuge tubes (amber and clear) 15 mL Falcon tubes Aluminum foil Ni-NTA agarose (QIAGEN, cat# 30310) Amicon Ultra-15 Centrifugal Filter, 10 kDa cutoff (Millipore-Sigma, cat# UFC901024) 10 mL Poly-Prep® Chromatography Column (Bio-Rad, cat#7311550) 12.5% Tris-Glycine SDS-PAGE gels 30 °C water bath 30 °C benchtop incubator TLX-Optima ultracentrifuge (Beckman Coulter Inc.) or equivalent TLA100 rotor (Beckman Coulter Inc.) or equivalent Gel dryer Storage phosphor screen (GE Healthcare Life Sciences) Typhoon Phosphorimager (GE Healthcare Life Sciences) Refrigerated benchtop swinging bucket centrifuge (Beckman Coulter Inc.) Note 1: All reagents and apparatus that come into contact with the IVT reaction should be nuclease-free following standard procedures . ..

Protease Inhibitor:

Article Title: Loss of the Notch effector RBPJ promotes tumorigenesis
Article Snippet: .. In brief, the cells were washed two times with PBS, resuspended in four pellet volumes of buffer A (10 mM Hepes-KOH, pH 7.8, 10 mM KCl, 1.5 mM MgCl2 , 5 mM with addition of a protease inhibitor cocktail; all from Sigma-Aldrich), pelleted for 15 s at 12,000 g and resuspended in 375 µl buffer A containing 0.5% NP-40 (Sigma-Aldrich). .. After mixing, cells were incubated at 4°C for 10 min to lyse the cells, centrifuged for 10 min at 12,000 g at 4°C, and the cytosolic extract was removed.

Article Title: A Novel Role of Lactosylceramide in the Regulation of Lipopolysaccharide/Interferon-γ-Mediated Inducible Nitric Oxide Synthase Gene Expression: Implications for Neuroinflammatory Diseases
Article Snippet: .. Cells were harvested, washed twice with ice-cold TBS, and lysed in 400 μl of buffer A containing (in m m ): 10 KCl, 2 MgCl2 , 0.5 dithiothreitol plus protease inhibitor mixture (Sigma) and 0.1% Nonidet P-40 in 10 m m HEPES, pH 7.9, for 10 min on ice. .. After centrifugation at 5000 × g for 10 min the pelleted nuclei were washed with buffer A without Nonidet P-40 and resuspended in 40 μl of buffer B containing 25% (v/v) glycerol, 0.42 m NaCl, and (in m m ) 1.5 MgCl2 , 0.2 EDTA, 0.5 dithiothreitol, and Complete protease inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, IN) in 20 m m HEPES, pH 7.9, for 30 min on ice.

Multiple Displacement Amplification:

Article Title: Loss of the Notch effector RBPJ promotes tumorigenesis
Article Snippet: Nuclear lysates were collected from shRandom, shRBPJ, and shRBPJ-set2 overexpressing MDA-MB-231 cells for the RBPJ EMSA assays. .. In brief, the cells were washed two times with PBS, resuspended in four pellet volumes of buffer A (10 mM Hepes-KOH, pH 7.8, 10 mM KCl, 1.5 mM MgCl2 , 5 mM with addition of a protease inhibitor cocktail; all from Sigma-Aldrich), pelleted for 15 s at 12,000 g and resuspended in 375 µl buffer A containing 0.5% NP-40 (Sigma-Aldrich).

Generated:

Article Title: The Cysteine-rich Domain of the DHHC3 Palmitoyltransferase Is Palmitoylated and Contains Tightly Bound Zinc *
Article Snippet: Elutions were pooled and rotated with ANTI-FLAG® M2-agarose affinity gel (Sigma) equilibrated with Buffer A (50 m m Tris, pH 7.4, 100 m m NaCl, 10% glycerol, 0.1% DDM, 1 m m EDTA, 5 μg/ml leupeptin, and 1 μ m pepstatin A) at 4 °C for 2 h. The FLAG affinity resin was washed five times with a total of 50 column volumes of Buffer A and eluted with 5 × 1 column volumes of Buffer A + 0.3 mg/ml FLAG peptide (Sigma). .. The concentration of enzyme was determined by plotting elution samples along a linear curve generated with known concentrations of bovine serum albumin stained with SYPRO® Ruby protein gel stain (Lonza, Rockland, ME) and quantified using a VersaDocTM 5000 imaging system.

Imaging:

Article Title: The Cysteine-rich Domain of the DHHC3 Palmitoyltransferase Is Palmitoylated and Contains Tightly Bound Zinc *
Article Snippet: Elutions were pooled and rotated with ANTI-FLAG® M2-agarose affinity gel (Sigma) equilibrated with Buffer A (50 m m Tris, pH 7.4, 100 m m NaCl, 10% glycerol, 0.1% DDM, 1 m m EDTA, 5 μg/ml leupeptin, and 1 μ m pepstatin A) at 4 °C for 2 h. The FLAG affinity resin was washed five times with a total of 50 column volumes of Buffer A and eluted with 5 × 1 column volumes of Buffer A + 0.3 mg/ml FLAG peptide (Sigma). .. The concentration of enzyme was determined by plotting elution samples along a linear curve generated with known concentrations of bovine serum albumin stained with SYPRO® Ruby protein gel stain (Lonza, Rockland, ME) and quantified using a VersaDocTM 5000 imaging system.

Sonication:

Article Title: Rad50 ATPase activity is regulated by DNA ends and requires coordination of both active sites
Article Snippet: Insect cells expressing complexes were lysed in Ni-A buffer (50 mM KH2 PO4 , 10% glycerol, 2.5 mM imidazole, 20 mM β-mercaptoethanol, 0.5 M KCl) using homogenization followed by sonication. .. The eluate was diluted with Buffer A (25 mM Tris pH 8.0, 100 mM NaCl, 10% glycerol, 1 mM DTT) to 150 mM NaCl, and applied to 1 ml anti-FLAG M2 antibody resin (Sigma), washed with Buffer A, with five volumes of 0.5 M LiCl and eluted with 0.1 mg/ml FLAG peptide (Sigma) in Buffer A. Aliquots were flash frozen and stored at −70°C.

Binding Assay:

Article Title: A Novel Role of Lactosylceramide in the Regulation of Lipopolysaccharide/Interferon-γ-Mediated Inducible Nitric Oxide Synthase Gene Expression: Implications for Neuroinflammatory Diseases
Article Snippet: Cells were harvested, washed twice with ice-cold TBS, and lysed in 400 μl of buffer A containing (in m m ): 10 KCl, 2 MgCl2 , 0.5 dithiothreitol plus protease inhibitor mixture (Sigma) and 0.1% Nonidet P-40 in 10 m m HEPES, pH 7.9, for 10 min on ice. .. Nuclear proteins (10 μg) were used for the electrophoretic mobility shift assay for the detection of NF-κB DNA binding activities.

Mutagenesis:

Article Title: Rad50 ATPase activity is regulated by DNA ends and requires coordination of both active sites
Article Snippet: Protein purification WT and mutant hMR and yMR, were purified as described previously ( ) with modifications. .. The eluate was diluted with Buffer A (25 mM Tris pH 8.0, 100 mM NaCl, 10% glycerol, 1 mM DTT) to 150 mM NaCl, and applied to 1 ml anti-FLAG M2 antibody resin (Sigma), washed with Buffer A, with five volumes of 0.5 M LiCl and eluted with 0.1 mg/ml FLAG peptide (Sigma) in Buffer A. Aliquots were flash frozen and stored at −70°C.

Purification:

Article Title: Identification of a functional docking site in the Rpn1 LRR domain for the UBA-UBL domain protein Ddi1
Article Snippet: Paragraph title: Purification of 26S proteasomes for immunoblotting ... Lysates were thawed in buffer A (50 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM MgCl2 , 5 mM ATP), bound to anti-Flag resin (Sigma, St. Louis, MO, U.S.), washed three times with buffer A supplemented with 0.2% Triton X-100, then washed two times with buffer B (25 mM Tris pH 7.5, 10 mM MgCl2 , 2 mM ATP) prior to elution with Flag Peptide (Sigma).

Article Title: Rad50 ATPase activity is regulated by DNA ends and requires coordination of both active sites
Article Snippet: The complexes of His6 -tagged Rad50 and FLAG-tagged Mre11 were purified using Ni-NTA, SP and anti-FLAG resin. .. The eluate was diluted with Buffer A (25 mM Tris pH 8.0, 100 mM NaCl, 10% glycerol, 1 mM DTT) to 150 mM NaCl, and applied to 1 ml anti-FLAG M2 antibody resin (Sigma), washed with Buffer A, with five volumes of 0.5 M LiCl and eluted with 0.1 mg/ml FLAG peptide (Sigma) in Buffer A. Aliquots were flash frozen and stored at −70°C.

Article Title: The Cysteine-rich Domain of the DHHC3 Palmitoyltransferase Is Palmitoylated and Contains Tightly Bound Zinc *
Article Snippet: Paragraph title: Two-step Purification of DHHC3 ... Elutions were pooled and rotated with ANTI-FLAG® M2-agarose affinity gel (Sigma) equilibrated with Buffer A (50 m m Tris, pH 7.4, 100 m m NaCl, 10% glycerol, 0.1% DDM, 1 m m EDTA, 5 μg/ml leupeptin, and 1 μ m pepstatin A) at 4 °C for 2 h. The FLAG affinity resin was washed five times with a total of 50 column volumes of Buffer A and eluted with 5 × 1 column volumes of Buffer A + 0.3 mg/ml FLAG peptide (Sigma).

Article Title: Transmembrane pores formed by syntheticp-octiphenyl ?-barrels with internal carboxylate clusters: Regulation of ion transport by pH and Mg2+- complexed 8-aminonaphthalene-1,3,6-trisulfonate
Article Snippet: .. A solution of EYPC (50 mg in 50 μl of EtOH; Avanti Polar Lipids) and sodium cholate (22.4 mg; Sigma) in 950 μl of buffer A was dialyzed against 50 ml of buffer A [ > 6 h, 5 mM N -[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES) (Sigma)/12.5 mM 8-aminonaphtalene-1,3,6-trisulfonate (ANTS)/45.0 mM p -xylenebis(pyridinium)bromide (DPX) (Molecular Probes)/20 mM KCl, pH 7.0 ( )] and 1,000 ml of buffer B [ > 12 h, 5 mM TES/100 mM KCl, pH 7.0 ( )], purified by gel filtration (Sephadex G-50, buffer B), and diluted to 6 ml. .. ANTS/DPX-efflux was recorded as a function of time by measuring changes in emission intensity It [λem = 510 nm, λex = 353 nm ( , ), FluoroMax-2; Jobin-Yvon-Spex, Longjumeau, France] after addition of these EYPC-SUVs (10 mM EYPC, 50 μl) to stirred thermostated buffer C (1.95 ml/10 mM Mes/100 mM KCl, pH 4.5–7.0) to give emission intensity I 0 .

Protein Purification:

Article Title: Rad50 ATPase activity is regulated by DNA ends and requires coordination of both active sites
Article Snippet: Paragraph title: Protein purification ... The eluate was diluted with Buffer A (25 mM Tris pH 8.0, 100 mM NaCl, 10% glycerol, 1 mM DTT) to 150 mM NaCl, and applied to 1 ml anti-FLAG M2 antibody resin (Sigma), washed with Buffer A, with five volumes of 0.5 M LiCl and eluted with 0.1 mg/ml FLAG peptide (Sigma) in Buffer A. Aliquots were flash frozen and stored at −70°C.

Lysis:

Article Title: The Cysteine-rich Domain of the DHHC3 Palmitoyltransferase Is Palmitoylated and Contains Tightly Bound Zinc *
Article Snippet: Cleared lysate was then passed three times through an equilibrated 0.5-ml column of Ni-NTA resin, washed with 40 column volumes of lysis buffer and 30 column volumes of lysis buffer containing 15 m m imidazole. .. Elutions were pooled and rotated with ANTI-FLAG® M2-agarose affinity gel (Sigma) equilibrated with Buffer A (50 m m Tris, pH 7.4, 100 m m NaCl, 10% glycerol, 0.1% DDM, 1 m m EDTA, 5 μg/ml leupeptin, and 1 μ m pepstatin A) at 4 °C for 2 h. The FLAG affinity resin was washed five times with a total of 50 column volumes of Buffer A and eluted with 5 × 1 column volumes of Buffer A + 0.3 mg/ml FLAG peptide (Sigma).

Article Title: Transmembrane pores formed by syntheticp-octiphenyl ?-barrels with internal carboxylate clusters: Regulation of ion transport by pH and Mg2+- complexed 8-aminonaphthalene-1,3,6-trisulfonate
Article Snippet: A solution of EYPC (50 mg in 50 μl of EtOH; Avanti Polar Lipids) and sodium cholate (22.4 mg; Sigma) in 950 μl of buffer A was dialyzed against 50 ml of buffer A [ > 6 h, 5 mM N -[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES) (Sigma)/12.5 mM 8-aminonaphtalene-1,3,6-trisulfonate (ANTS)/45.0 mM p -xylenebis(pyridinium)bromide (DPX) (Molecular Probes)/20 mM KCl, pH 7.0 ( )] and 1,000 ml of buffer B [ > 12 h, 5 mM TES/100 mM KCl, pH 7.0 ( )], purified by gel filtration (Sephadex G-50, buffer B), and diluted to 6 ml. .. Lysis (100%) was determined with Triton X-100 (10% aqueous, 50 μl) to give emission intensity I ∞ .

Chloramphenicol Acetyltransferase Assay:

Article Title: In vitro assays for targeting and insertion of tail-anchored proteins into the ER membrane
Article Snippet: .. E. coli S30 extract containing tRNACUA Opt ( ; ; ) 250 µM RF-1 aptamer ( ) 40–80 µM Tagless T7 RNA polymerase ( ) 500–600 µM Tagless CmRS(D286R) ( ) 150–200 µM His6 -tagged Sgt2 ( ) Small molecule mix (SMM) (see Recipe) 10 mM amino acid mix (−Met or +Met) (see Recipe) 40 mM 7-hydroxycoumaryl ethylglycine (Cm) (Millipore-Sigma, cat# 792551) Plasmid encoding protein of interest (POI), such as a model TA (SUMO-Bos1-opsin) under control of the T7 promoter (pUC19-Strep3 -SUMO-Bos1-opsin or pUC19-Strep3 -SUMO-Amb-Bos1-opsin) ( ) 11 mCi/mL 35 S-Met (Perkin Elmer, catNEG009T001MC) Buffer A (see Recipe) Fixing solution (see Recipe) 3M Potassium acetate (KOAc) dissolved in Buffer A (Millipore-Sigma, cat#P5708) 2M Imidazole dissolved in Buffer A (pH~7.0) (Millipore-Sigma, cat#I5513) Maxi-prep kit (QIAGEN, cat#12963) sterile 100×15mm petri dish 0.6 mL microfuge tubes (amber and clear) 15 mL Falcon tubes Aluminum foil Ni-NTA agarose (QIAGEN, cat# 30310) Amicon Ultra-15 Centrifugal Filter, 10 kDa cutoff (Millipore-Sigma, cat# UFC901024) 10 mL Poly-Prep® Chromatography Column (Bio-Rad, cat#7311550) 12.5% Tris-Glycine SDS-PAGE gels 30 °C water bath 30 °C benchtop incubator TLX-Optima ultracentrifuge (Beckman Coulter Inc.) or equivalent TLA100 rotor (Beckman Coulter Inc.) or equivalent Gel dryer Storage phosphor screen (GE Healthcare Life Sciences) Typhoon Phosphorimager (GE Healthcare Life Sciences) Refrigerated benchtop swinging bucket centrifuge (Beckman Coulter Inc.) Note 1: All reagents and apparatus that come into contact with the IVT reaction should be nuclease-free following standard procedures . ..

Article Title: In vitro assays for targeting and insertion of tail-anchored proteins into the ER membrane
Article Snippet: .. E. coli S30 extract containing tRNACUA Opt ( ; ; ) 250 µM RF-1 aptamer ( ) 40–80 µM Tagless T7 RNA polymerase ( ) 500–600 µM Tagless CmRS(D286R) ( ) 150–200 µM His6 -tagged Sgt2 ( ) Small molecule mix (SMM) (see Recipe) 10 mM amino acid mix (−Met or +Met) (see Recipe) 40 mM 7-hydroxycoumaryl ethylglycine (Cm) (Millipore-Sigma, cat# 792551) Plasmid encoding protein of interest (POI), such as a model TA (SUMO-Bos1-opsin) under control of the T7 promoter (pUC19-Strep3 -SUMO-Bos1-opsin or pUC19-Strep3 -SUMO-Amb-Bos1-opsin) ( ) 11 mCi/mL 35 S-Met (Perkin Elmer, catNEG009T001MC) Buffer A (see Recipe) Fixing solution (see Recipe) 3M Potassium acetate (KOAc) dissolved in Buffer A (Millipore-Sigma, cat#P5708) 2M Imidazole dissolved in Buffer A (pH~7.0) (Millipore-Sigma, cat#I5513) Maxi-prep kit (QIAGEN, cat#12963) sterile 100×15mm petri dish 0.6 mL microfuge tubes (amber and clear) 15 mL Falcon tubes Aluminum foil Ni-NTA agarose (QIAGEN, cat# 30310) Amicon Ultra-15 Centrifugal Filter, 10 kDa cutoff (Millipore-Sigma, cat# UFC901024) 10 mL Poly-Prep® Chromatography Column (Bio-Rad, cat#7311550) 12.5% Tris-Glycine SDS-PAGE gels 30 °C water bath 30 °C benchtop incubator TLX-Optima ultracentrifuge (Beckman Coulter Inc.) or equivalent TLA100 rotor (Beckman Coulter Inc.) or equivalent Gel dryer Storage phosphor screen (GE Healthcare Life Sciences) Typhoon Phosphorimager (GE Healthcare Life Sciences) Refrigerated benchtop swinging bucket centrifuge (Beckman Coulter Inc.) Note 1: All reagents and apparatus that come into contact with the IVT reaction should be nuclease-free following standard procedures . ..

SDS Page:

Article Title: In vitro assays for targeting and insertion of tail-anchored proteins into the ER membrane
Article Snippet: .. E. coli S30 extract containing tRNACUA Opt ( ; ; ) 250 µM RF-1 aptamer ( ) 40–80 µM Tagless T7 RNA polymerase ( ) 500–600 µM Tagless CmRS(D286R) ( ) 150–200 µM His6 -tagged Sgt2 ( ) Small molecule mix (SMM) (see Recipe) 10 mM amino acid mix (−Met or +Met) (see Recipe) 40 mM 7-hydroxycoumaryl ethylglycine (Cm) (Millipore-Sigma, cat# 792551) Plasmid encoding protein of interest (POI), such as a model TA (SUMO-Bos1-opsin) under control of the T7 promoter (pUC19-Strep3 -SUMO-Bos1-opsin or pUC19-Strep3 -SUMO-Amb-Bos1-opsin) ( ) 11 mCi/mL 35 S-Met (Perkin Elmer, cat#NEG009T001MC) Buffer A (see Recipe) Fixing solution (see Recipe) 3M Potassium acetate (KOAc) dissolved in Buffer A (Millipore-Sigma, cat#P5708) 2M Imidazole dissolved in Buffer A (pH~7.0) (Millipore-Sigma, cat#I5513) Maxi-prep kit (QIAGEN, cat#12963) sterile 100×15mm petri dish 0.6 mL microfuge tubes (amber and clear) 15 mL Falcon tubes Aluminum foil Ni-NTA agarose (QIAGEN, cat# 30310) Amicon Ultra-15 Centrifugal Filter, 10 kDa cutoff (Millipore-Sigma, cat# UFC901024) 10 mL Poly-Prep® Chromatography Column (Bio-Rad, cat#7311550) 12.5% Tris-Glycine SDS-PAGE gels 30 °C water bath 30 °C benchtop incubator TLX-Optima ultracentrifuge (Beckman Coulter Inc.) or equivalent TLA100 rotor (Beckman Coulter Inc.) or equivalent Gel dryer Storage phosphor screen (GE Healthcare Life Sciences) Typhoon Phosphorimager (GE Healthcare Life Sciences) Refrigerated benchtop swinging bucket centrifuge (Beckman Coulter Inc.) Note 1: All reagents and apparatus that come into contact with the IVT reaction should be nuclease-free following standard procedures . ..

Article Title: In vitro assays for targeting and insertion of tail-anchored proteins into the ER membrane
Article Snippet: .. E. coli S30 extract containing tRNACUA Opt ( ; ; ) 250 µM RF-1 aptamer ( ) 40–80 µM Tagless T7 RNA polymerase ( ) 500–600 µM Tagless CmRS(D286R) ( ) 150–200 µM His6 -tagged Sgt2 ( ) Small molecule mix (SMM) (see Recipe) 10 mM amino acid mix (−Met or +Met) (see Recipe) 40 mM 7-hydroxycoumaryl ethylglycine (Cm) (Millipore-Sigma, cat# 792551) Plasmid encoding protein of interest (POI), such as a model TA (SUMO-Bos1-opsin) under control of the T7 promoter (pUC19-Strep3 -SUMO-Bos1-opsin or pUC19-Strep3 -SUMO-Amb-Bos1-opsin) ( ) 11 mCi/mL 35 S-Met (Perkin Elmer, cat#NEG009T001MC) Buffer A (see Recipe) Fixing solution (see Recipe) 3M Potassium acetate (KOAc) dissolved in Buffer A (Millipore-Sigma, cat#P5708) 2M Imidazole dissolved in Buffer A (pH~7.0) (Millipore-Sigma, cat#I5513) Maxi-prep kit (QIAGEN, cat#12963) sterile 100×15mm petri dish 0.6 mL microfuge tubes (amber and clear) 15 mL Falcon tubes Aluminum foil Ni-NTA agarose (QIAGEN, cat# 30310) Amicon Ultra-15 Centrifugal Filter, 10 kDa cutoff (Millipore-Sigma, cat# UFC901024) 10 mL Poly-Prep® Chromatography Column (Bio-Rad, cat#7311550) 12.5% Tris-Glycine SDS-PAGE gels 30 °C water bath 30 °C benchtop incubator TLX-Optima ultracentrifuge (Beckman Coulter Inc.) or equivalent TLA100 rotor (Beckman Coulter Inc.) or equivalent Gel dryer Storage phosphor screen (GE Healthcare Life Sciences) Typhoon Phosphorimager (GE Healthcare Life Sciences) Refrigerated benchtop swinging bucket centrifuge (Beckman Coulter Inc.) Note 1: All reagents and apparatus that come into contact with the IVT reaction should be nuclease-free following standard procedures . ..

Plasmid Preparation:

Article Title: In vitro assays for targeting and insertion of tail-anchored proteins into the ER membrane
Article Snippet: .. E. coli S30 extract containing tRNACUA Opt ( ; ; ) 250 µM RF-1 aptamer ( ) 40–80 µM Tagless T7 RNA polymerase ( ) 500–600 µM Tagless CmRS(D286R) ( ) 150–200 µM His6 -tagged Sgt2 ( ) Small molecule mix (SMM) (see Recipe) 10 mM amino acid mix (−Met or +Met) (see Recipe) 40 mM 7-hydroxycoumaryl ethylglycine (Cm) (Millipore-Sigma, cat# 792551) Plasmid encoding protein of interest (POI), such as a model TA (SUMO-Bos1-opsin) under control of the T7 promoter (pUC19-Strep3 -SUMO-Bos1-opsin or pUC19-Strep3 -SUMO-Amb-Bos1-opsin) ( ) 11 mCi/mL 35 S-Met (Perkin Elmer, cat#NEG009T001MC) Buffer A (see Recipe) Fixing solution (see Recipe) 3M Potassium acetate (KOAc) dissolved in Buffer A (Millipore-Sigma, cat#P5708) 2M Imidazole dissolved in Buffer A (pH~7.0) (Millipore-Sigma, cat#I5513) Maxi-prep kit (QIAGEN, cat#12963) sterile 100×15mm petri dish 0.6 mL microfuge tubes (amber and clear) 15 mL Falcon tubes Aluminum foil Ni-NTA agarose (QIAGEN, cat# 30310) Amicon Ultra-15 Centrifugal Filter, 10 kDa cutoff (Millipore-Sigma, cat# UFC901024) 10 mL Poly-Prep® Chromatography Column (Bio-Rad, cat#7311550) 12.5% Tris-Glycine SDS-PAGE gels 30 °C water bath 30 °C benchtop incubator TLX-Optima ultracentrifuge (Beckman Coulter Inc.) or equivalent TLA100 rotor (Beckman Coulter Inc.) or equivalent Gel dryer Storage phosphor screen (GE Healthcare Life Sciences) Typhoon Phosphorimager (GE Healthcare Life Sciences) Refrigerated benchtop swinging bucket centrifuge (Beckman Coulter Inc.) Note 1: All reagents and apparatus that come into contact with the IVT reaction should be nuclease-free following standard procedures . ..

Article Title: In vitro assays for targeting and insertion of tail-anchored proteins into the ER membrane
Article Snippet: .. E. coli S30 extract containing tRNACUA Opt ( ; ; ) 250 µM RF-1 aptamer ( ) 40–80 µM Tagless T7 RNA polymerase ( ) 500–600 µM Tagless CmRS(D286R) ( ) 150–200 µM His6 -tagged Sgt2 ( ) Small molecule mix (SMM) (see Recipe) 10 mM amino acid mix (−Met or +Met) (see Recipe) 40 mM 7-hydroxycoumaryl ethylglycine (Cm) (Millipore-Sigma, cat# 792551) Plasmid encoding protein of interest (POI), such as a model TA (SUMO-Bos1-opsin) under control of the T7 promoter (pUC19-Strep3 -SUMO-Bos1-opsin or pUC19-Strep3 -SUMO-Amb-Bos1-opsin) ( ) 11 mCi/mL 35 S-Met (Perkin Elmer, cat#NEG009T001MC) Buffer A (see Recipe) Fixing solution (see Recipe) 3M Potassium acetate (KOAc) dissolved in Buffer A (Millipore-Sigma, cat#P5708) 2M Imidazole dissolved in Buffer A (pH~7.0) (Millipore-Sigma, cat#I5513) Maxi-prep kit (QIAGEN, cat#12963) sterile 100×15mm petri dish 0.6 mL microfuge tubes (amber and clear) 15 mL Falcon tubes Aluminum foil Ni-NTA agarose (QIAGEN, cat# 30310) Amicon Ultra-15 Centrifugal Filter, 10 kDa cutoff (Millipore-Sigma, cat# UFC901024) 10 mL Poly-Prep® Chromatography Column (Bio-Rad, cat#7311550) 12.5% Tris-Glycine SDS-PAGE gels 30 °C water bath 30 °C benchtop incubator TLX-Optima ultracentrifuge (Beckman Coulter Inc.) or equivalent TLA100 rotor (Beckman Coulter Inc.) or equivalent Gel dryer Storage phosphor screen (GE Healthcare Life Sciences) Typhoon Phosphorimager (GE Healthcare Life Sciences) Refrigerated benchtop swinging bucket centrifuge (Beckman Coulter Inc.) Note 1: All reagents and apparatus that come into contact with the IVT reaction should be nuclease-free following standard procedures . ..

Homogenization:

Article Title: Rad50 ATPase activity is regulated by DNA ends and requires coordination of both active sites
Article Snippet: Insect cells expressing complexes were lysed in Ni-A buffer (50 mM KH2 PO4 , 10% glycerol, 2.5 mM imidazole, 20 mM β-mercaptoethanol, 0.5 M KCl) using homogenization followed by sonication. .. The eluate was diluted with Buffer A (25 mM Tris pH 8.0, 100 mM NaCl, 10% glycerol, 1 mM DTT) to 150 mM NaCl, and applied to 1 ml anti-FLAG M2 antibody resin (Sigma), washed with Buffer A, with five volumes of 0.5 M LiCl and eluted with 0.1 mg/ml FLAG peptide (Sigma) in Buffer A. Aliquots were flash frozen and stored at −70°C.

Concentration Assay:

Article Title: The Cysteine-rich Domain of the DHHC3 Palmitoyltransferase Is Palmitoylated and Contains Tightly Bound Zinc *
Article Snippet: Elutions were pooled and rotated with ANTI-FLAG® M2-agarose affinity gel (Sigma) equilibrated with Buffer A (50 m m Tris, pH 7.4, 100 m m NaCl, 10% glycerol, 0.1% DDM, 1 m m EDTA, 5 μg/ml leupeptin, and 1 μ m pepstatin A) at 4 °C for 2 h. The FLAG affinity resin was washed five times with a total of 50 column volumes of Buffer A and eluted with 5 × 1 column volumes of Buffer A + 0.3 mg/ml FLAG peptide (Sigma). .. Elutions were pooled and rotated with ANTI-FLAG® M2-agarose affinity gel (Sigma) equilibrated with Buffer A (50 m m Tris, pH 7.4, 100 m m NaCl, 10% glycerol, 0.1% DDM, 1 m m EDTA, 5 μg/ml leupeptin, and 1 μ m pepstatin A) at 4 °C for 2 h. The FLAG affinity resin was washed five times with a total of 50 column volumes of Buffer A and eluted with 5 × 1 column volumes of Buffer A + 0.3 mg/ml FLAG peptide (Sigma).

Staining:

Article Title: The Cysteine-rich Domain of the DHHC3 Palmitoyltransferase Is Palmitoylated and Contains Tightly Bound Zinc *
Article Snippet: Elutions were pooled and rotated with ANTI-FLAG® M2-agarose affinity gel (Sigma) equilibrated with Buffer A (50 m m Tris, pH 7.4, 100 m m NaCl, 10% glycerol, 0.1% DDM, 1 m m EDTA, 5 μg/ml leupeptin, and 1 μ m pepstatin A) at 4 °C for 2 h. The FLAG affinity resin was washed five times with a total of 50 column volumes of Buffer A and eluted with 5 × 1 column volumes of Buffer A + 0.3 mg/ml FLAG peptide (Sigma). .. The concentration of enzyme was determined by plotting elution samples along a linear curve generated with known concentrations of bovine serum albumin stained with SYPRO® Ruby protein gel stain (Lonza, Rockland, ME) and quantified using a VersaDocTM 5000 imaging system.

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  • 95
    Millipore hat assay buffer
    Inhibitory effects of TA on <t>HAT</t> activity. (A) A colorimetric assay was performed to measure HAT activity in the presence of TA at the indicated concentrations. The results are presented as percentages relative to the control sample, which was incubated without TA (left panel). The IC 50 value of TA inhibition of HAT activity was calculated using Prism software (right panel). (B) An in vitro HAT assay using autoradiography was performed to evaluate the effect of the anti-HAT activity of TA on the acetylation of a synthetic H4 peptide by a <t>HeLa</t> NE. The arrow indicates histone H4 (left panel). The autoradiography was quantified using Image J software (right panel). The values presented are the means ± SD of two independent experiments. Means with different superscript letters are significantly different, p
    Hat Assay Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hat assay buffer/product/Millipore
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    99
    Millipore sds loading buffer
    RanBP3L inhibits BMP-induced transcriptional responses. (A) Schematic diagram of RanBP3L and RanBP3. RanBP3L contains a Ran-binding domain (RanBD) with 71% identity to that of RanBP3. (B) RanBP3L binds with RanGTP. HEK293T cells were cotransfected with Myc-RanBP3L and FLAG-RanQ69L. RanBP3L was immunoprecipitated (IP) with anti-Myc antibody and then subjected to <t>SDS-PAGE</t> and Western blotting to detect RanBP3L-bound RanQ69L. WCL, whole-cell lysate. (C to E) RanBP3L inhibits BMP2-induced Id1-luc reporter activity. C3H10T1/2 (C), C2C12 (D), and ATDC5 cells (E) were transfected with RanBP3L, PPM1A, or RanBP3 together with Id1-luc reporter plasmids. BMP2 treatment and luciferase assays were done as described in Materials and Methods. RLU, relative light units. (F) RanBP3L does not affect TGF-β-induced SBE-luc reporter activity in HaCaT cells. HaCaT cells were transfected with RanBP3 or RanBP3L together with the SBE-luc reporter plasmid and then treated with 2 ng/ml TGF-β for 12 h. The experiment was carried out as described above for panels C to E. (G and H) Stable expression of hRanBP3L in C2C12 cells decreases mRNA levels of Id1 (G) and Id2 (H). Cells were treated with LDN193189 (BMP type I receptor inhibitor) (LDN) or BMP2 (50 ng/ml) for 6 h, and total RNA was extracted for qRT-PCR analysis. (I) RanBP3L knockdown efficacy was measured in C2C12 cells. C2C12 cells were transfected with RanBP3L siRNAs or control siRNA (siCTR), and total RNA was extracted for qRT-PCR analysis. (J) Knockdown of RanBP3L enhances BMP2-induced Id1-luc reporter activity. C2C12 cells were transfected with RanBP3L siRNAs or control siRNA, together with the Id1-luc reporter plasmid. (K and L) Knockdown of RanBP3L increases Id1 mRNA expression (K) and Id2 mRNA expression (L). C2C12 cells were transfected with the indicated siRNAs and treated with BMP2 (50 ng/ml) for 6 h or not treated.
    Sds Loading Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 172 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore co immunoprecipitation co ip buffer
    Effect of HSD10-modification on CypD and how it may influence cancer cell growth and death. A. EV and HSD10 ov whole cell lysates were analyzed for CypD protein expression using immunoblotting. β-actin was used as the loading control, and CypD was normalized to actin (n = 4). B. Control shRNA and HSD10 shRNA whole cell lysates were analyzed for CypD protein expression using immunoblotting. β-actin was used as the loading control, and CypD was normalized to actin (n = 4). C. EV and HSD10 ov whole cell lysates were analyzed for HSD10-CypD complexes using <t>co-immunoprecipitation.</t> β-actin was used as the loading control for the input. The immunoblots demonstrate an increased HSD10-CypD interaction in PC-12 cells overexpressing HSD10 compared to EV cells. D. Confocal microscopy demonstrating immunofluorescence staining of HSD10 alone (red), CypD alone (green), and these two antigens co-localized (yellow) in EV and HSD10 ov cells. E. Immunofluorescence staining of HSD10 alone (red), mitochondrial marker SODII alone (green), and these two antigens co-localized (yellow) in HSD10 ov cells. F. Immunofluorescence staining of CypD alone (green), mitochondrial marker Hsp60 alone (red), and these two antigens co-localized (yellow) in HSD10 ov cells. Scale bar in F : 20 μm. G-H. Quantification of HSD10 and CypD fluorescence densities (depicted in D ) displayed as fold increase (n = 4). Data presented as mean ± SE. *P
    Co Immunoprecipitation Co Ip Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millipore buffer c human smp30 gnl
    Overall structure of mouse <t>SMP30/GNL.</t> The structure is shown as a rainbow colored cartoon with N-terminus = blue and C-terminus = red. The divalent metal ion (labeled as M 2+ ) located at the center of the structure is shown as an orange sphere.
    Buffer C Human Smp30 Gnl, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Inhibitory effects of TA on HAT activity. (A) A colorimetric assay was performed to measure HAT activity in the presence of TA at the indicated concentrations. The results are presented as percentages relative to the control sample, which was incubated without TA (left panel). The IC 50 value of TA inhibition of HAT activity was calculated using Prism software (right panel). (B) An in vitro HAT assay using autoradiography was performed to evaluate the effect of the anti-HAT activity of TA on the acetylation of a synthetic H4 peptide by a HeLa NE. The arrow indicates histone H4 (left panel). The autoradiography was quantified using Image J software (right panel). The values presented are the means ± SD of two independent experiments. Means with different superscript letters are significantly different, p

    Journal: Molecular Metabolism

    Article Title: Tannic acid, a novel histone acetyltransferase inhibitor, prevents non-alcoholic fatty liver disease both in vivo and in vitro model

    doi: 10.1016/j.molmet.2018.11.001

    Figure Lengend Snippet: Inhibitory effects of TA on HAT activity. (A) A colorimetric assay was performed to measure HAT activity in the presence of TA at the indicated concentrations. The results are presented as percentages relative to the control sample, which was incubated without TA (left panel). The IC 50 value of TA inhibition of HAT activity was calculated using Prism software (right panel). (B) An in vitro HAT assay using autoradiography was performed to evaluate the effect of the anti-HAT activity of TA on the acetylation of a synthetic H4 peptide by a HeLa NE. The arrow indicates histone H4 (left panel). The autoradiography was quantified using Image J software (right panel). The values presented are the means ± SD of two independent experiments. Means with different superscript letters are significantly different, p

    Article Snippet: For autoradiography-based in vitro HAT activity assays, HeLa cell nuclear extract was incubated with HAT assay buffer (50 mM HEPES, pH 8.0; 10% glycerol; 1 mM DTT; 1 mM phenylmethylsulfonyl fluoride; and 10 mM sodium butyrate), 1 μL [3 H] acetyl-CoA, and 5 μg of biotinylated-H4 peptide (Millipore, Billerica, MA, USA) along with TA at the indicated concentrations at 30 °C for 1 h. The reactions were stopped by adding 5 × sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer and samples were separated on 15% SDS–PAGE gels, followed by autoradiographic analysis.

    Techniques: HAT Assay, Activity Assay, Colorimetric Assay, Incubation, Inhibition, Software, In Vitro, Autoradiography

    RanBP3L inhibits BMP-induced transcriptional responses. (A) Schematic diagram of RanBP3L and RanBP3. RanBP3L contains a Ran-binding domain (RanBD) with 71% identity to that of RanBP3. (B) RanBP3L binds with RanGTP. HEK293T cells were cotransfected with Myc-RanBP3L and FLAG-RanQ69L. RanBP3L was immunoprecipitated (IP) with anti-Myc antibody and then subjected to SDS-PAGE and Western blotting to detect RanBP3L-bound RanQ69L. WCL, whole-cell lysate. (C to E) RanBP3L inhibits BMP2-induced Id1-luc reporter activity. C3H10T1/2 (C), C2C12 (D), and ATDC5 cells (E) were transfected with RanBP3L, PPM1A, or RanBP3 together with Id1-luc reporter plasmids. BMP2 treatment and luciferase assays were done as described in Materials and Methods. RLU, relative light units. (F) RanBP3L does not affect TGF-β-induced SBE-luc reporter activity in HaCaT cells. HaCaT cells were transfected with RanBP3 or RanBP3L together with the SBE-luc reporter plasmid and then treated with 2 ng/ml TGF-β for 12 h. The experiment was carried out as described above for panels C to E. (G and H) Stable expression of hRanBP3L in C2C12 cells decreases mRNA levels of Id1 (G) and Id2 (H). Cells were treated with LDN193189 (BMP type I receptor inhibitor) (LDN) or BMP2 (50 ng/ml) for 6 h, and total RNA was extracted for qRT-PCR analysis. (I) RanBP3L knockdown efficacy was measured in C2C12 cells. C2C12 cells were transfected with RanBP3L siRNAs or control siRNA (siCTR), and total RNA was extracted for qRT-PCR analysis. (J) Knockdown of RanBP3L enhances BMP2-induced Id1-luc reporter activity. C2C12 cells were transfected with RanBP3L siRNAs or control siRNA, together with the Id1-luc reporter plasmid. (K and L) Knockdown of RanBP3L increases Id1 mRNA expression (K) and Id2 mRNA expression (L). C2C12 cells were transfected with the indicated siRNAs and treated with BMP2 (50 ng/ml) for 6 h or not treated.

    Journal: Molecular and Cellular Biology

    Article Title: Nuclear Export of Smads by RanBP3L Regulates Bone Morphogenetic Protein Signaling and Mesenchymal Stem Cell Differentiation

    doi: 10.1128/MCB.00121-15

    Figure Lengend Snippet: RanBP3L inhibits BMP-induced transcriptional responses. (A) Schematic diagram of RanBP3L and RanBP3. RanBP3L contains a Ran-binding domain (RanBD) with 71% identity to that of RanBP3. (B) RanBP3L binds with RanGTP. HEK293T cells were cotransfected with Myc-RanBP3L and FLAG-RanQ69L. RanBP3L was immunoprecipitated (IP) with anti-Myc antibody and then subjected to SDS-PAGE and Western blotting to detect RanBP3L-bound RanQ69L. WCL, whole-cell lysate. (C to E) RanBP3L inhibits BMP2-induced Id1-luc reporter activity. C3H10T1/2 (C), C2C12 (D), and ATDC5 cells (E) were transfected with RanBP3L, PPM1A, or RanBP3 together with Id1-luc reporter plasmids. BMP2 treatment and luciferase assays were done as described in Materials and Methods. RLU, relative light units. (F) RanBP3L does not affect TGF-β-induced SBE-luc reporter activity in HaCaT cells. HaCaT cells were transfected with RanBP3 or RanBP3L together with the SBE-luc reporter plasmid and then treated with 2 ng/ml TGF-β for 12 h. The experiment was carried out as described above for panels C to E. (G and H) Stable expression of hRanBP3L in C2C12 cells decreases mRNA levels of Id1 (G) and Id2 (H). Cells were treated with LDN193189 (BMP type I receptor inhibitor) (LDN) or BMP2 (50 ng/ml) for 6 h, and total RNA was extracted for qRT-PCR analysis. (I) RanBP3L knockdown efficacy was measured in C2C12 cells. C2C12 cells were transfected with RanBP3L siRNAs or control siRNA (siCTR), and total RNA was extracted for qRT-PCR analysis. (J) Knockdown of RanBP3L enhances BMP2-induced Id1-luc reporter activity. C2C12 cells were transfected with RanBP3L siRNAs or control siRNA, together with the Id1-luc reporter plasmid. (K and L) Knockdown of RanBP3L increases Id1 mRNA expression (K) and Id2 mRNA expression (L). C2C12 cells were transfected with the indicated siRNAs and treated with BMP2 (50 ng/ml) for 6 h or not treated.

    Article Snippet: After several washes, precipitated proteins were eluted in SDS loading buffer, separated by SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore), and detected by Western blotting with appropriate antibodies.

    Techniques: Binding Assay, Immunoprecipitation, SDS Page, Western Blot, Activity Assay, Transfection, Luciferase, Plasmid Preparation, Expressing, Quantitative RT-PCR

    Effect of HSD10-modification on CypD and how it may influence cancer cell growth and death. A. EV and HSD10 ov whole cell lysates were analyzed for CypD protein expression using immunoblotting. β-actin was used as the loading control, and CypD was normalized to actin (n = 4). B. Control shRNA and HSD10 shRNA whole cell lysates were analyzed for CypD protein expression using immunoblotting. β-actin was used as the loading control, and CypD was normalized to actin (n = 4). C. EV and HSD10 ov whole cell lysates were analyzed for HSD10-CypD complexes using co-immunoprecipitation. β-actin was used as the loading control for the input. The immunoblots demonstrate an increased HSD10-CypD interaction in PC-12 cells overexpressing HSD10 compared to EV cells. D. Confocal microscopy demonstrating immunofluorescence staining of HSD10 alone (red), CypD alone (green), and these two antigens co-localized (yellow) in EV and HSD10 ov cells. E. Immunofluorescence staining of HSD10 alone (red), mitochondrial marker SODII alone (green), and these two antigens co-localized (yellow) in HSD10 ov cells. F. Immunofluorescence staining of CypD alone (green), mitochondrial marker Hsp60 alone (red), and these two antigens co-localized (yellow) in HSD10 ov cells. Scale bar in F : 20 μm. G-H. Quantification of HSD10 and CypD fluorescence densities (depicted in D ) displayed as fold increase (n = 4). Data presented as mean ± SE. *P

    Journal: BMC Cancer

    Article Title: Overexpression of 17β-hydroxysteroid dehydrogenase type 10 increases pheochromocytoma cell growth and resistance to cell death

    doi: 10.1186/s12885-015-1173-5

    Figure Lengend Snippet: Effect of HSD10-modification on CypD and how it may influence cancer cell growth and death. A. EV and HSD10 ov whole cell lysates were analyzed for CypD protein expression using immunoblotting. β-actin was used as the loading control, and CypD was normalized to actin (n = 4). B. Control shRNA and HSD10 shRNA whole cell lysates were analyzed for CypD protein expression using immunoblotting. β-actin was used as the loading control, and CypD was normalized to actin (n = 4). C. EV and HSD10 ov whole cell lysates were analyzed for HSD10-CypD complexes using co-immunoprecipitation. β-actin was used as the loading control for the input. The immunoblots demonstrate an increased HSD10-CypD interaction in PC-12 cells overexpressing HSD10 compared to EV cells. D. Confocal microscopy demonstrating immunofluorescence staining of HSD10 alone (red), CypD alone (green), and these two antigens co-localized (yellow) in EV and HSD10 ov cells. E. Immunofluorescence staining of HSD10 alone (red), mitochondrial marker SODII alone (green), and these two antigens co-localized (yellow) in HSD10 ov cells. F. Immunofluorescence staining of CypD alone (green), mitochondrial marker Hsp60 alone (red), and these two antigens co-localized (yellow) in HSD10 ov cells. Scale bar in F : 20 μm. G-H. Quantification of HSD10 and CypD fluorescence densities (depicted in D ) displayed as fold increase (n = 4). Data presented as mean ± SE. *P

    Article Snippet: Cells were washed twice with pre-chilled PBS, and then harvested, centrifuged, and suspended in 250 μl Co-Immunoprecipitation (Co-IP) buffer containing 150 mM NaCl, 50 mM Tris–HCl, pH 7.4, 1 mM EDTA, 0.5% NP-40, and 100X protease inhibitor (EMD Millipore).

    Techniques: Modification, Expressing, shRNA, Immunoprecipitation, Western Blot, Confocal Microscopy, Immunofluorescence, Staining, Marker, Fluorescence

    Overall structure of mouse SMP30/GNL. The structure is shown as a rainbow colored cartoon with N-terminus = blue and C-terminus = red. The divalent metal ion (labeled as M 2+ ) located at the center of the structure is shown as an orange sphere.

    Journal: PLoS ONE

    Article Title: Structural Basis of the ?-Lactone-Ring Formation in Ascorbic Acid Biosynthesis by the Senescence Marker Protein-30/Gluconolactonase

    doi: 10.1371/journal.pone.0053706

    Figure Lengend Snippet: Overall structure of mouse SMP30/GNL. The structure is shown as a rainbow colored cartoon with N-terminus = blue and C-terminus = red. The divalent metal ion (labeled as M 2+ ) located at the center of the structure is shown as an orange sphere.

    Article Snippet: The obtained sample was applied to a Phenyl-Sepharose CL-4B column (SIGMA-ALDRICH, 50 ml) pre-equilibrated with buffer C. Human SMP30/GNL was eluted by a gradient from 0% to 100% of buffer D. The eluted fractions were concentrated using an Amicon Ultra-15 centrifugal filter unit (MWCO 10,000) (Millipore) and further purified using a HiLoad 16/60 Superdex 200 pg column (GE Healthcare, 120 ml) pre-equilibrated with buffer E. The eluted fractions were dialyzed against buffer D. Purified proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie brilliant blue (CBB) stain ( ).

    Techniques: Labeling

    Active site structures of mouse and human SMP30/GNL. ( A ) Mouse SMP30/GNL in the substrate-free form, ( B ) the mouse SMP30/GNL–1,5-AG complex, ( C ) the human SMP30/GNL–1,5-AG complex, ( D ) the mouse SMP30/GNL– d -glucose complex, and ( E ) the mouse SMP30/GNL–xylitol complex. Lid loop residues of mouse SMP30/GNL and human SMP30/GNL are shown in purple and blue, respectively. Carbon atoms of ligand residues for the divalent metal ion (orange sphere) and those for substrate/product analogues are shown in green and yellow, respectively. Other carbon atoms are shown in white.

    Journal: PLoS ONE

    Article Title: Structural Basis of the ?-Lactone-Ring Formation in Ascorbic Acid Biosynthesis by the Senescence Marker Protein-30/Gluconolactonase

    doi: 10.1371/journal.pone.0053706

    Figure Lengend Snippet: Active site structures of mouse and human SMP30/GNL. ( A ) Mouse SMP30/GNL in the substrate-free form, ( B ) the mouse SMP30/GNL–1,5-AG complex, ( C ) the human SMP30/GNL–1,5-AG complex, ( D ) the mouse SMP30/GNL– d -glucose complex, and ( E ) the mouse SMP30/GNL–xylitol complex. Lid loop residues of mouse SMP30/GNL and human SMP30/GNL are shown in purple and blue, respectively. Carbon atoms of ligand residues for the divalent metal ion (orange sphere) and those for substrate/product analogues are shown in green and yellow, respectively. Other carbon atoms are shown in white.

    Article Snippet: The obtained sample was applied to a Phenyl-Sepharose CL-4B column (SIGMA-ALDRICH, 50 ml) pre-equilibrated with buffer C. Human SMP30/GNL was eluted by a gradient from 0% to 100% of buffer D. The eluted fractions were concentrated using an Amicon Ultra-15 centrifugal filter unit (MWCO 10,000) (Millipore) and further purified using a HiLoad 16/60 Superdex 200 pg column (GE Healthcare, 120 ml) pre-equilibrated with buffer E. The eluted fractions were dialyzed against buffer D. Purified proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie brilliant blue (CBB) stain ( ).

    Techniques:

    Catalytic reaction of mouse SMP30/GNL. ( A ) The γ-lactone-forming reaction catalyzed by mouse SMP30/GNL. The product of the catalytic reaction of SMP30/GNL is l -gulono-γ-lactone, which is in turn converted to ascorbic acid by gluconolactone oxidase. ( B–D ) Substrate and product analogues used in this study: ( B ) xylitol, ( C ) 1,5-anhydro- d -glucitol (1,5-AG), and ( D ) d -glucose. Corresponding atoms in these molecules are marked in light orange and light yellow.

    Journal: PLoS ONE

    Article Title: Structural Basis of the ?-Lactone-Ring Formation in Ascorbic Acid Biosynthesis by the Senescence Marker Protein-30/Gluconolactonase

    doi: 10.1371/journal.pone.0053706

    Figure Lengend Snippet: Catalytic reaction of mouse SMP30/GNL. ( A ) The γ-lactone-forming reaction catalyzed by mouse SMP30/GNL. The product of the catalytic reaction of SMP30/GNL is l -gulono-γ-lactone, which is in turn converted to ascorbic acid by gluconolactone oxidase. ( B–D ) Substrate and product analogues used in this study: ( B ) xylitol, ( C ) 1,5-anhydro- d -glucitol (1,5-AG), and ( D ) d -glucose. Corresponding atoms in these molecules are marked in light orange and light yellow.

    Article Snippet: The obtained sample was applied to a Phenyl-Sepharose CL-4B column (SIGMA-ALDRICH, 50 ml) pre-equilibrated with buffer C. Human SMP30/GNL was eluted by a gradient from 0% to 100% of buffer D. The eluted fractions were concentrated using an Amicon Ultra-15 centrifugal filter unit (MWCO 10,000) (Millipore) and further purified using a HiLoad 16/60 Superdex 200 pg column (GE Healthcare, 120 ml) pre-equilibrated with buffer E. The eluted fractions were dialyzed against buffer D. Purified proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie brilliant blue (CBB) stain ( ).

    Techniques:

    Top view of the substrate-binding cavity. Surface representation of ( A ) mouse SMP30/GNL, ( B ) DFPase, ( C ) Drp35, and ( D ) PON. Residues in the lid loop of mouse SMP30/GNL and the divalent metal ions (labeled as M 2+ ) are shown in purple and orange, respectively. Structures of DFPase, Drp35, and PON are superposed onto mouse SMP30/GNL by the SSM fitting using the program Superpose [27] in the CCP4 program suite [20] , and all molecules are viewed from the same direction.

    Journal: PLoS ONE

    Article Title: Structural Basis of the ?-Lactone-Ring Formation in Ascorbic Acid Biosynthesis by the Senescence Marker Protein-30/Gluconolactonase

    doi: 10.1371/journal.pone.0053706

    Figure Lengend Snippet: Top view of the substrate-binding cavity. Surface representation of ( A ) mouse SMP30/GNL, ( B ) DFPase, ( C ) Drp35, and ( D ) PON. Residues in the lid loop of mouse SMP30/GNL and the divalent metal ions (labeled as M 2+ ) are shown in purple and orange, respectively. Structures of DFPase, Drp35, and PON are superposed onto mouse SMP30/GNL by the SSM fitting using the program Superpose [27] in the CCP4 program suite [20] , and all molecules are viewed from the same direction.

    Article Snippet: The obtained sample was applied to a Phenyl-Sepharose CL-4B column (SIGMA-ALDRICH, 50 ml) pre-equilibrated with buffer C. Human SMP30/GNL was eluted by a gradient from 0% to 100% of buffer D. The eluted fractions were concentrated using an Amicon Ultra-15 centrifugal filter unit (MWCO 10,000) (Millipore) and further purified using a HiLoad 16/60 Superdex 200 pg column (GE Healthcare, 120 ml) pre-equilibrated with buffer E. The eluted fractions were dialyzed against buffer D. Purified proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie brilliant blue (CBB) stain ( ).

    Techniques: Binding Assay, Labeling

    Proposed catalytic reaction mechanism of mouse SMP30/GNL. The divalent metal ion in the active site is labeled as M 2+ .

    Journal: PLoS ONE

    Article Title: Structural Basis of the ?-Lactone-Ring Formation in Ascorbic Acid Biosynthesis by the Senescence Marker Protein-30/Gluconolactonase

    doi: 10.1371/journal.pone.0053706

    Figure Lengend Snippet: Proposed catalytic reaction mechanism of mouse SMP30/GNL. The divalent metal ion in the active site is labeled as M 2+ .

    Article Snippet: The obtained sample was applied to a Phenyl-Sepharose CL-4B column (SIGMA-ALDRICH, 50 ml) pre-equilibrated with buffer C. Human SMP30/GNL was eluted by a gradient from 0% to 100% of buffer D. The eluted fractions were concentrated using an Amicon Ultra-15 centrifugal filter unit (MWCO 10,000) (Millipore) and further purified using a HiLoad 16/60 Superdex 200 pg column (GE Healthcare, 120 ml) pre-equilibrated with buffer E. The eluted fractions were dialyzed against buffer D. Purified proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie brilliant blue (CBB) stain ( ).

    Techniques: Labeling

    Structural comparison of the lid loops of mouse and human SMP30/GNL. ( A ) Lid loops of mouse and human SMP30/GNL in the substrate free form are shown in purple and blue, respectively. The divalent metal ion (labeled as M 2+ ) is shown in orange. ( B, C ) SA-omit maps (mFo-DFc maps) for the lid loop residues in mouse ( B ) and human ( C ) SMP30/GNL. The contour levels of the SA-omit maps are 3.0 σ and 2.0 σ for panels B and C, respectively. ( D ) Surface representation of mouse SMP30/GNL around the lid loop. The entrance for the substrate-binding cavity is indicated by an arrow. Residues in the lid loop are shown in purple.

    Journal: PLoS ONE

    Article Title: Structural Basis of the ?-Lactone-Ring Formation in Ascorbic Acid Biosynthesis by the Senescence Marker Protein-30/Gluconolactonase

    doi: 10.1371/journal.pone.0053706

    Figure Lengend Snippet: Structural comparison of the lid loops of mouse and human SMP30/GNL. ( A ) Lid loops of mouse and human SMP30/GNL in the substrate free form are shown in purple and blue, respectively. The divalent metal ion (labeled as M 2+ ) is shown in orange. ( B, C ) SA-omit maps (mFo-DFc maps) for the lid loop residues in mouse ( B ) and human ( C ) SMP30/GNL. The contour levels of the SA-omit maps are 3.0 σ and 2.0 σ for panels B and C, respectively. ( D ) Surface representation of mouse SMP30/GNL around the lid loop. The entrance for the substrate-binding cavity is indicated by an arrow. Residues in the lid loop are shown in purple.

    Article Snippet: The obtained sample was applied to a Phenyl-Sepharose CL-4B column (SIGMA-ALDRICH, 50 ml) pre-equilibrated with buffer C. Human SMP30/GNL was eluted by a gradient from 0% to 100% of buffer D. The eluted fractions were concentrated using an Amicon Ultra-15 centrifugal filter unit (MWCO 10,000) (Millipore) and further purified using a HiLoad 16/60 Superdex 200 pg column (GE Healthcare, 120 ml) pre-equilibrated with buffer E. The eluted fractions were dialyzed against buffer D. Purified proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie brilliant blue (CBB) stain ( ).

    Techniques: Labeling, Binding Assay