Cells were grown on glass coverslips, washed with phosphate-buffered saline (PBS), fixed with 1 or 4% paraformaldehyde (in PBS) for 10 min, and permeabilized with 0.2% Triton X-100 in PBS for 5 min. After blocking with 2% bovine serum albumin (BSA) in PBS for at least 1 h, cells were incubated with primary antibodies, diluted in 2% BSA in PBS for 1 h at room temperature (RT) or overnight at 4°C, washed three times with PBS, and incubated with secondary antibodies in 2% BSA in PBS for 1 h at RT. After immunostaining, cells were washed with PBS and mounted on microscopic slides with VectaShield (VectorLabs) for microscopic analysis. The following primary antibodies were used in this study: anti-calnexin (Abcam, ab22595), anti-calreticulin (ThermoScientific, PA3-900), CREST (ImmunoVision, HCT0100), anti–Ki-67 (Abcam, ab16667), anti-KIF4 (Abcam, ab3815), anti-KIF22 (Abcam, ab222187), anti-LBR (abcam, ab32535), mAb414 (Abcam, ab24609), anti-MAD2 (Bethyl Laboratories, A300-301A), anti-topoisomeraseII (Abcam, ab109524), anti–α-tubulin (Sigma, T5168), and anti–γ-H2AX (Millipore, 05-636-l). The antibody against human SUN1 has been previously described (Sosa et al., 2013 (link)). For secondary antibodies, Alexa Fluor–conjugated antibodies (goat, 1:300; Life Technologies) were used.
T5168
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany, France, Canada, Spain
The T5168 is a piece of laboratory equipment manufactured by Merck Group. It is designed for a specific core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach. Therefore, the description for this product is not available.
Automatically generated - may contain errors
Lab products found in correlation
α tubulin, by Merck Group (9 mentions)
Anti α tubulin, by Merck Group (8 mentions)
Ab1791, by Abcam (7 mentions)
Mouse anti α tubulin, by Merck Group (5 mentions)
F3165, by Merck Group (5 mentions)
A1978, by Merck Group (5 mentions)
T3559, by Merck Group (5 mentions)
T6557, by Merck Group (5 mentions)
Nitrocellulose membrane, by Bio-Rad (5 mentions)
Polyvinylidene difluoride membrane, by Merck Group (5 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Mab374, by Merck Group (4 mentions)
F7425, by Merck Group (4 mentions)
Amersham imager 600, by GE Healthcare (4 mentions)
Ab8898, by Abcam (4 mentions)
F1804, by Merck Group (4 mentions)
Ab290, by Abcam (4 mentions)
T6793, by Merck Group (4 mentions)
Odyssey infrared imaging system, by LI COR (4 mentions)
212 protocols using t5168
1
Immunostaining of Cellular Components
2
Comprehensive Immunodetection Protocol for Neurodegenerative Markers
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For western blot analysis rabbit anti-rab10 (Cell Signaling, 8127S, 1:1000), rabbit anti-phospho-rab10 (Abcam, ab230261, 1:1000), rabbit anti-rab8 (Cell signaling, 6971S, 1:1000), mouse anti-β-3-tubulin (Cell signaling, 4466S, 1:4000), rabbit anti-TH (Millipore, 657012, 1:4000), rabbit anti pS129 aSyn (Abcam, ab51253, 1:1000), mouse anti alpha synuclein (BD Biosciences, 610787, 1:2000), rabbit anti-LRRK2 (Abcam, ab133474, 1:500), rabbit anti-phospho S935 LRRK2 (Abcam, ab133450, 1:500). rabbit anti-alpha synuclein (Santacruz, SC7011-R, 1:2000), rabbit anti-Glucocerebrosidase (Sigma, G4171), mouse anti-alpha tubulin (Sigma, T5168, 1:40,000), mouse anti-GAPDH (Millipore, MAB374, 1:5000). The secondary antibodies used in western blot analysis goat anti-mouse and goat anti-rabbit (Jackson ImmunoResearch lab, #115–035–146, #111–035–144, 1:10,000). For immunocytochemistry, rabbit anti-pS129 aSyn (Abcam, ab51253, 1:200), mouse anti-β-3-tubulin (BioLegend, 801202, 1:500), rabbit anti-β-3-tubulin (BioLegend, 802001, 1:500), sheep anti-TH (Novus, NB300–110, 1:500) mouse anti-MAP2 (Sigma, M4403, 1:500).
3
Antibody Immunofluorescence and Western Blot Analysis
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IF primary: rabbit anti-CENP-A (Active Motif, #39713, 1:1000); guinea pig anti-CENP-C31 (link) (1:1000); rabbit anti-ATPsyn-α (Abcam #ab151229, 1:100); mouse anti-ATPsyn-β (Abcam #ab14730, 1:200); goat anti-ATPsyn-γ (Abcam #ab190310, 1:200); rat anti-mCherry (Chromotek 5F8 1:500); mouse anti-tubulin (Sigma DM1A, 1:100); guinea pig anti-MEI-S33232 (link) (gift from T. Orr-Weaver 1:500) and rabbit anti-GFP (Santa Cruz SC-8334). Guinea pig anti-ATPsyn-βlike antibodies (1:200) were generated by co-injection of two KLH and BSA conjugated peptides CKTDAELVKKKDE (amino acid 68–80) and GDAPPAKAEAKKDEK (amino acid 575–587) marked on Supplementary Fig. 1D . IF secondary: Alexa-488, -546, -647-coupled goat anti-mouse, goat anti-rabbit or goat anti-guinea pig (Life Technologies, 1:500). Western analysis: rabbit anti-CENP-A (Active Motif, #39713, 1:1000), mouse anti-ATPsyn-α (Abcam #ab14748, 1:1000), mouse anti-ATPsyn-β (Abcam #14730, 1:1000), rat anti-GST (Chromotek 6G9, 1:1000), mouse anti-red (Chromotek 6G6, 1:1000), mouse anti-poly-his (Sigma Aldrich #H1029, 1:1000), mouse anti-tubulin (Sigma Aldrich #T5168, 1:10,000) and rabbit anti-histone 3 (Millipore 17–10,254; 1:50,000); guinea pig anti-ATPsyn-βlike (1:1000). Uncropped scans of blots are provided in Supplementary Figure 5 .
4
Antibody Usage in Protein Detection
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We used the following antibodies: A300-516A (Thermo Fisher), polyclonal anti-DNA-PKcs 1:5000; EPR2302 (GeneTex, Inc), monoclonal anti-FANCD2 1:2000; T5168 (Merck), monoclonal anti-αTubulin 1:8000; sc73614 (Santa Cruz Biotechnology), monoclonal anti-Vinculin 1:1000; A0545 (Merk), HRP (horseradish peroxidase)-conjugated anti-rabbit IgG 1:10 000; A0168 (Merk), HRP-conjugated anti-mouse IgG 1:10 000.
5
Western Blot Analysis of Protein Samples
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Total cell lysates were boiled for 10 min at 99°C in 1× Laemmli buffer supplemented with 4.2% (vol/vol) β-mercaptoethanol. Proteins were separated by electrophoresis on precast 4 to 20% SDS-PAGE gels (Bio-Rad Laboratories) and semidry blotted on nitrocellulose membranes. Membranes were blocked with a 5% (wt/vol) lowfat milk solution in PBS. Proteins were detected with the following primary antibodies: rabbit anti-Flag (F7425; Sigma-Aldrich), rabbit anti-HA (ab9110; Abcam), and rabbit anti-MED25 (NBP2-55868; Novus Biologicals). As a control for equal loading, we used mouse anti-α-tubulin (T5168; Merck) or rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) (ab9485; Abcam). We used the following secondary antibodies: streptavidin-horseradish peroxidase (HRP) (RPN1231; Amersham Life Science), ECL donkey anti-rabbit IgG–HRP (NA934V, GE Healthcare), and ECL sheep anti-mouse IgG–HRP (NA931V; GE Healthcare). Protein bands were visualized by using Pierce ECL Western blotting substrate (32106; Thermo Fisher Scientific) and a chemiluminescence imager (Amersham Imager 600; GE Healthcare).
6
Western Blot Analysis of MAGEA4 Protein
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Cellular, vesicular, or purified protein samples were suspended in Laemmli buffer and denatured for 10 min at 100°. The lysates were separated electrophoretically using 10% SDS-PAGE gel and blotted onto a PVDF membrane using Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad Laboratories; Hercules, CA, USA). Affinity-purified rabbit polyclonal antibodies against MAGEA4 (2.5 mg/mL) [53 (link)] were used for immunoblotting at dilutions of 1:10,000. Alpha-tubulin (dilution 1:4000; T5168; Merck; St. Louis, MO, USA), anti- TSG101 (dilution 1:10,000, T5701; Merck; St. Louis, MO, USA), anti-E2Tag antibody 5E11 (dilution 1:10,000; Icosagen; Tartu, Estonia) were used in different experiments. Goat anti-rabbit (1 mg/mL, LabAS; Tartu, Estonia) and goat anti-mouse (1 mg/mL, LabAS) antibodies conjugated with HRP were used as secondary antibodies at a dilution of 1:10,000. Protein signals were detected using ECL Western blotting (GE Healthcare; Marlborough, MA USA) reagents. The staining of SDS-PAGE gels was performed with PageBlue Protein Staining Solution (Thermo Scientific; Waltham, MA, USA).
7
Antibody Generation and Characterization
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Primary antibodies against the following proteins were used: rabbit anti-RNP-8 (Kim et al. 2009 (link)); mouse anti-NPC (Mab414, Covance), anti-tubulin (T5168, Sigma), and anti-GLD-2 (Millonigg et al. 2014 (link)); and rat anti-GLD-3 (Eckmann et al. 2002 (link)). The mouse anti-GLD-2 (Millonigg et al. 2014 (link)) recognized the C terminus of the protein. The rabbit anti-GLD-2 antibody used in the immunoprecipitation experiments was generated by immunizing New Zealand white rabbits with an epitope that covered amino acid 959–1113 of GLD-2, fused to a GST-affinity tag. The fusion protein had been expressed in BL21 bacteria and purified via a GST column to homogeneity. An analogously prepared MBP-fusion protein had been coupled to Hi-TRAP NHS columns (GE Healthcare) and used for affinity purification of the immune serum rb184.
8
Protein Expression and Analysis Protocol
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Whole cell lysates were prepared and separated using SDS‐PAGE gel electrophoresis at 125 volts (V) for 1.5 h, before transfer at 100 V for 1 h, as previously described.19 Membranes were blocked before being probed overnight at 4°C with primary antibodies against Cx43, β‐catenin (Cell Signaling Technology D10A8), N‐cadherin, fibronectin, collagen I (Abcam ab34710), (all at 1:1000) and α‐tubulin (Sigma‐Aldrich T5168), at a dilution of 1:20,000, as a house‐keeping protein. After washing, membranes were probed with secondary antibody (goat anti‐rabbit 800 and/or goat anti‐mouse) at 1:20,000 for 1 h at RT. Bands were visualised using an Odyssey Fc imaging unit before semi‐quantification and analysis using ImageStudioLite software (5.2.5).
9
Chromatin Regulation Protocols in Cell Lines
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Eco-Pack 2–293 cells (Clontech) and 293T cells (gifted from Dr. Yao [Ito et al., 2001 (link)]) were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal calf serum. Wild-type and Suv39h dn iMEFs (Lachner et al., 2001 (link)) were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal calf serum, 0.1 mM beta-mercaptoethanol, and 1x non-essential amino acids. The following antibodies were used in this study: anti-FLAG-M2 (F3165: Sigma-Aldrich, RRID:AB_259529 ), anti-FLAG-M2-HRP (A8592: Sigma-Aldrich, RRID: AB_439702 ), anti-α-tubulin (T5168: Sigma, RRID: AB_477579 ), anti-Suv39h1 (8729: Cell Signaling, RRID: AB_10829612 ), anti-H3 (ab21054: Abcam, RRID: AB_880437 ), anti-H3K9me3 (ab8898: Abcam, RRID: AB_306848 and 2F3 (RRID: AB_2616099 )(Chandra et al., 2012 (link)), anti-HP1β (BMP002: MBL, RRID: AB_843158 ), and anti-GST (27-4577-01: GE Healthcare, RRID: AB_771432 ).
10
Western Blot Analysis of Cellular Proteins
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Proteins were separated by SDS-PAGE in 10% gels and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with PVDF Blocking Reagent for Can Get Signal (Toyobo, Osaka, Japan) for 1 h. The membranes were incubated with primary antibodies against mouse GCAT (1:1,000; sc-86466, Santa Cruz Biotechnology, Dallas, TX, USA), mouse SHMT2 (1:1,000; #12762, Cell Signaling Technology, Danvers, MA, USA), β-ACTIN (1:10,000; A1978, Sigma, St. Louis, MO, USA) or α-TUBULIN (1:50,000; T5168, Sigma, St. Louis, MO, USA) for 1 h at room temperature; Can Get Signal Immunoreaction Enhancer Solution 1 (Toyobo) was used for dilution. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies against goat IgG (1:20,000; HAF109, R&D Systems, Minneapolis, MN, USA), rabbit IgG (1:10,000; G-21234, Thermo Fisher Scientific, Waltham, MA,USA) or mouse IgG (1:10,000; G-21040, Life Technologies, Carlsbad, CA, USA) for 1 h at room temperature; Can Get Signal Immunoreaction Enhancer Solution 2 (Toyobo) was used for dilution. Bands were detected with a bio-imaging analyser, EZ-Capture ST (ATTO, Tokyo, Japan) using ECL Select Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK).
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