Cells were resuspended in lysis buffer (50 mM Tris-HCl pH 7.4, 130 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing protease inhibitors (Complete, Roche Applied Science, Indianapolis) and protein concentration was measured using the Bradford assay (#5000006, BioRad, Hercules, CA, USA). Protein samples (30 μg/lane) were subjected to SDS-PAGE and then transferred to PVDF membranes (Immobilon-PSQ PVDF Membrane, Merck Millipore, Darmstadt, Germany). After blocking, membranes were incubated with primary antibodies against the following proteins: α-tubulin (1:5000, T6199, Sigma-Aldrich, St. Louis, MO, USA), acetylated α-tubulin (1:1000, T7451-25UL, Sigma-Aldrich, St. Louis, MO, USA), and β-actin (1:10,000, A5441, Sigma-Aldrich, St. Louis, MO, USA). β-actin was used as the loading control. Goat Anti-Mouse IgG (H+L) DryLightTM 680 Conjugated (1:10,000, 35518, Invitrogen, Waltham, MA, USA) was used as the secondary antibody. Immunoblots were incubated for 1 h at room temperature and developed using Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). Protein expression levels were calculated using ImageJ Software (ImageJ2, version 2.3.0/1.53q).
T6199
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany
The T6199 is a laboratory equipment product manufactured by Merck Group. It serves as a general-purpose instrument for various scientific applications. The core function of the T6199 is to perform accurate measurements and analyses in a laboratory setting. No further details or interpretations about the intended use of this product are provided.
Automatically generated - may contain errors
Lab products found in correlation
F1804, by Merck Group (6 mentions)
α tubulin, by Merck Group (6 mentions)
Ab1791, by Abcam (5 mentions)
Sc 11415, by Santa Cruz Biotechnology (5 mentions)
Hoechst 33342, by Thermo Fisher Scientific (5 mentions)
Odyssey infrared imaging system, by LI COR (4 mentions)
F3165, by Merck Group (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Anti α tubulin, by Merck Group (4 mentions)
A5441, by Merck Group (3 mentions)
Nitrocellulose membrane, by Merck Group (3 mentions)
Sap7f407, by Enzo Life Sciences (3 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (3 mentions)
T5326, by Merck Group (3 mentions)
A 21244, by Thermo Fisher Scientific (3 mentions)
L9393, by Merck Group (3 mentions)
Ab110413, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Odyssey clx, by LI COR (3 mentions)
143 protocols using t6199
1
Quantifying Protein Expression via Western Blot
2
Worm Protein Extraction and Western Blot
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Worms were lysed in protein lysis buffer (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 0.25% sodium deoxycholate, 1 mM EDTA and protease inhibitor (Roche)) using a Precellys 24 homogenizer. Worm lysates were centrifuged at 10,000 rpm for 10 min at 4 °C and the supernatant was collected. Protein concentrations were determined with standard BCA protein assay (Thermoscientific). 30 μg of total protein was separated by SDS-PAGE, transferred to nitrocellulose membranes (Millipore) and subjected to immunoblotting. Western blot analysis was performed with anti-PGES2 antibody (Bioss, bs-2639R, 1:500; RRID:AB_10860215) and α-tubulin (Sigma, T6199, 1:5,000; RRID:AB_477583).
3
Western Blot Analysis of Cellular Proteins
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Protein samples were extracted from cells and mitochondria, respectively, using RIPA lysis buffer (Chromotek) supplemented with protease inhibitors. Protein samples were diluted in LDS sample buffer (4×, Invitrogen) supplemented with sample reducing agent (10×, Invitrogen). Samples were heated at 95̊C for 5 min before loading on a 4–12% gradient Bis-Tris gel (Invitrogen). MOPS SDS running buffer (Invitrogen) was used and supplied with antioxidants (Invitrogen). The gel was soaked in running buffer and run at 160 V for 75 min. The gel was transferred to a 0.45 µm nitrocellulose membrane (Cytiva) and protein at 30 V for 150 min, 4̊C. The membrane was blocked for 1 h in 5% skimmed milk (Sigma) in PBS-Tween 20 (0.1%, Sigma). The membrane was probed for anti-GFP (ab183734, abcam), anti-mCherry (1C51, ab125096, abcam), anti-VDAC1 (ab15895, abcam) and anti-α tubulin (DM1α, T6199, Sigma), diluted 1 : 10 000, 1 : 3000, 1 : 1500, 1 : 1500, respectively, in 5% milk (PBS-Tween). Primary antibody signal was detected using goat anti-mouse IgG conjugated to IRDye 680RD (ab216776, abcam) and goat anti-rabbit IgG conjugated to IRDye 800CW (ab216773, abcam) secondary antibodies, diluted 1 : 15 000, imaged with an Odyssey CLx imaging system (LI-CO Biosciences).
4
Immunoblotting with Specific Antibodies
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The following antibodies were used at the dilutions indicated: Anti-Tubulin (1:10,000, T6199, Sigma-Aldrich), Anti-RFP (1:700, 6G9, Chromotek) and Anti-NUF2 (1:100, sc-271-251, Santa Cruz).
5
Quantifying AMPA Receptor Subunits
6
Protein Extraction and Western Blot Analysis
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Cells were lysed in modified radioimmunoprecipitation assay buffer (modified RIPA) [50-mM Tris-HCl (pH 7.4), 150-mM NaCl, 1-mM EDTA, 1% NP-40, 0.25% sodium deoxycholate, 1-mM dithiothreitol, 1-mM phenylmethylsulfonyl fluoride, aprotinin (1 mg/ml), and leupeptin (1 mg/ml)]. Specific antibodies against α-tubulin (T6199, Sigma), CEBPD (sc-636, Santa Cruz Biotechnology), phospho-p44/42 (#4377, Cell Signaling), total p44/42 (#9102, Cell Signaling), phospho-p38 (#9211, Cell Signaling), total p38 MAPK (#9212, Cell Signaling), phospho-AKT (GTX61708, GeneTex), total AKT (GTX121937, GeneTex), SDF4 (10517-1-AP, Proteintech), and CXCR4 (60042-1- Ig, Proteintech) were used for western blotting.
7
EHEC Infection of Caco-2 Cells
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First, ~750 synchronized late L4 to young adult stage animals were plated on NGM plates seeded with EHEC strain EDL933 or control plates seeded with E. coli OP50 for 4 days at 20 °C. Polyclonal antibody to GFP (Abcam, ab6556), monoclonal antibody to SUN-1 (phospho-S43) (University of Vienna, Austria), and monoclonal antibody to α-tubulin (Sigma-Aldrich, T6199) were used for detection, Caco-2. In brief, 3 × 105 cells were seeded to a 10-cm cell culture dish. When monolayer cells became 70–80% confluent 4–6 days after the inoculum, they were infected by EHEC strain EDL933 at MOI (500:1) for 0.5 h. Monoclonal antibody to GAPDH (Abcam, ab181602), polyclonal antibody to histone H3 (phospho-S10) (Abcam, ab47297), monoclonal antibody to DIAPH1 (Cell Signaling, 14634), polyclonal antibody to DIAPH2 (Cell Signaling, 5474), polyclonal antibody to DIAPH3 (Sigma-Aldrich, SAB1409850), and monoclonal antibody to polyclonal phospho-PP1α (Thr320) (Cell Signaling, 2581) were used for detection.
8
Polyclonal Antibody Production Protocol
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For production of polyclonal antibodies, glutathione S-transferase (GST)-fusion proteins containing the specific antigenic regions of seven candidate proteins were expressed in Escherichia coli BL21 and affinity purified with glutathione Sepharose 4B (GE Healthcare). The recombinant proteins were used as antigens for producing rabbit polyclonal antisera. The antibodies were affinity purified using the appropriate proteins and an AminoLink immobilization kit (Pierce). The following commercially available antibodies were also used: a mouse monoclonal antibody against ADAM2 (1/1000, MAB19292) from Millipore; an antibody against α-tubulin (1/1000, T6199) from Sigma-Aldrich; and an antibody against GAPDH (1/1000, MCA4739) from Bio-Rad. As secondary antibodies for Western blot analysis, we used horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG (Jackson ImmunoResearch).
9
Cell Culture and Antibody Reagents
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HEK293T cells, L cells (an immortalized mouse fibroblast cell line), SW480 cells and HCT116 cells were obtained from Shanghai Life Academy of Sciences cell library (Shanghai, China). All cells were grown in DMEM medium (Invitrogen, Carlsbad, CA), and maintained in culture supplemented with 10% heat-inactivated fetal calf serum, 100 ug ml−1 of penicillin, and 100 μg ml−1 of streptomycin at 37 °C with 5% CO2 in a humidified incubator (Thermo, Waltham, MA). During the study, all cell cultures were periodically tested for mycoplasma by using MycoAlert Mycoplasma Detection Kit (Lonza, Rockland, ME). For western blot, antibodies specific for VGLL4 (1:500, ab140290), TEAD4 (1:500, ab58310), Histone H3 (1:1,000, ab6002) and β-catenin (1:1,000, ab2365) were purchased from Abcam (Cambridge, UK); those for FLAG (1:5,000, M4439) and α-tubulin (1:2,000, T6199) were from Sigma (St. Louis, MO); and those for TCF4 (1:500, sc-166699), c-Jun (1:1,000, sc-4113) and GST (1:1,000, sc-138) were bought from Santa Cruz Biotechnology (Santa Cruz, CA).
10
Western Blot Analysis of Cellular Proteins
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Cells were plated at 300,000 cells per well in 6-well plates and grown to confluence. Media was then removed, cells were washed twice with PBS, and 200 μL of RIPA lysis buffer containing sigma protease inhibitor cocktail (P8340, 1:100) was added to each well. Cells were incubated in lysis buffer for 10 min at RT then scrapped and collected. Lysates were analyzed by BCA and normalized for total protein content with lysis buffer then denatured in Laemmli loading buffer with 0.01 M DTT at 100 °C for 10 min. 10 μg of protein from each sample was then loaded onto Biorad Mini-PROTEAN® TGX™ 4–15% gels and run @ 150V for 55 min then transferred to Immobilon PVDF membrane at 35 V for 1 h. The membrane was blocked with 5% non-fat milk in TBS-T for 1 h at rt then incubated with the indicated antibodies in 2.5% non-fat milk overnight at 4 °C. The blot was then washed three-times with TBS-T and once with PBS-T before incubating with Licor IRDye secondary antibodies in Odyssey blocking buffer (#927-40000) + 0.2% Tween-20 and 0.01% Sodium dodecyl sulfate (SDS). The blot was washed 3 times with PBS-T for 5 min and once with PBS then imaged for fluorescent signal on an Odessey Classic Infrared Imaging System. Ferritin antibodies were obtained from Abcam (Ab75973) and tubulin antibodies were obtained from Sigma (T6199).
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