40ug of protein was loaded onto a 4–12% SDS-PAGE gradient gel (Bio-Rad, Hercules, CA, USA and Novex, Life Technologies, Grand Island, NY, USA) and resolved. Proteins were transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA), and blocked with Li-cor blocking buffer (Li-cor, Lincoln, NE, USA) for 1 hr at room temperature. Membranes were probed with primary antibody overnight at 4°C (mouse anti-TSP-1 1:250 (Abcam, Cambridge, MA, USA), mouse anti-TSP-2 1:250 (BD, San Jose, CA, USA), goat anti-TSP-4 1:500 (R&D Systems, Minneapolis, MN, USA), mouse anti-α2δ-1 1:500 (Sigma-Aldrich), and the next day probed with secondary antibody 1:20k (Li-cor) for 1 hr at room temperature. Membranes were dried for 2 hrs and scanned using Li-cor Odyssey Fc. Membranes were rehydrated in tris buffered saline (TBS) (50mM Tris, 150mM NaCl, pH 7.4) for 2 hours at room temperature. Membranes were then re-probed with mouse anti-tubulin 1:1000 (Abcam, Cambridge, MA, USA). The optical densities of all bands were quantified with Li-cor Image Studio and adjusted to within lane background subtraction using the standard Li-cor Image Studio-defined parameters. Bands of interest (1 lane per independent brain sample) were adjusted to the tubulin loading control values. n=6–8/treatment group.
Mouse anti tubulin
Manufactured by Abcam
Sourced in United States, United Kingdom
Mouse anti-tubulin is a primary antibody that detects the tubulin protein in mouse samples. Tubulin is a key structural component of the cytoskeleton in eukaryotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti slc31a1 ctr1, by Abcam (2 mentions)
Mouse anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Mouse anti myc, by Santa Cruz Biotechnology (2 mentions)
Mouse anti flag m2, by Merck Group (2 mentions)
Mouse anti rna pol 2, by Abcam (2 mentions)
Radioimmunoprecipitation assay buffer, by Merck Group (2 mentions)
Rabbit anti dyskerin, by Santa Cruz Biotechnology (2 mentions)
Mouse anti tcab1, by Merck Group (2 mentions)
Rabbit anti gar1, by Novus Biologicals (2 mentions)
Rabbit anti nhp2, by Proteintech (2 mentions)
Rabbit anti nop10, by Abcam (2 mentions)
Mouse anti strep, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Blocking buffer, by LI COR (1 mentions)
Goat anti tsp 4, by R&D Systems (1 mentions)
Mouse anti α2δ 1, by Merck Group (1 mentions)
Mouse anti tsp 1, by Abcam (1 mentions)
Mouse anti tsp 2, by BD (1 mentions)
25 protocols using mouse anti tubulin
1
Western Blot Analysis of Thrombospondin Proteins
2
AHA-Labeling and Biotin Enrichment of Newly Synthesized Proteins
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To assess the effect of 4E-BP overexpression, 4E-BPLLAA was induced in S2 cells stably transfected with pMT-4E-BPLLAA with 0.5mM copper sulfate for 2 h in methionine-free Schneider’s medium to enable AHA uptake. The cells were then labeled with 4 mM AHA (Anaspec) for 1 h. AHA-labeled proteins in the extracts were clicked to biotin-PEG4 alkyne (Invitrogen) using the manufacturer’s protocols. AHA-conjugated peptides were further enriched by incubation with Streptavidin-agarose (Thermo Fisher Scientific) followed by rigorous washing with PBS-Tween (0.5%). The bound fraction was collected by boiling the beads in sample buffer and analyzed by Western blotting with Streptavidin-IRDye 800 (Rockland Immunochemicals), guinea pig anti-BiP (1:1,000), mouse anti-Hsp70 (Abcam), mouse antiactin (EMD Millipore), and mouse antitubulin (Abcam).
3
Circadian Rhythms in Drosophila CRY
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Three day old cry-H [CS] or cry-L [CS] flies were placed in plastic vials (10x2 cm) containing sugar food (4.6% sugar, 4.6% brewer’s yeast, 1.25% agar, 0.2% methyl 4-hydroxybenzoate). Vials were placed in incubator for four days at 25°C in LD 12∶12. During the fourth day, flies were collected every 3 h beginning at ZT1 (one hour after lights on) and immediately frozen in liquid nitrogen. Proteins were isolated from 40–50 heads from each time point as in Edery et al. 1994 [22] (link). The protein preparation were separated in 8% polyacrylamide gels (37.5∶1, acrylamide:bisacrylamide ratio; Sigma Aldrich-USA). Blots were incubated with a CRY antiserum raised against N terminal residues 1–188 (1∶1000, [48] ) and mouse anti-tubulin (1∶5000, abcam-UK) diluted in TBST-5% dry milk. An anti- guinea pig IgG-HRP (1∶8000; Amersham-UK) and an anti-mouse IgG-HRP (1∶2000; Sigma Aldrich-USA) were used as secondary antibodies. Visualization of CRY and TUB was performed by chemiluminescence (ECL Advance Western Blotting Detection Kit, GE HEALTHCARE) and autoradiography. Three blots for each line were analysed using Image J software (version 1.45b) for the quantification of the signals. Background signal was subtracted, normalised by the TUB signal, and expressed as % of maximum signal in each plot.
4
Extracellular Vesicle Protein Profiling
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The samples used for identification of widely expressed protein markers in EVs and quantification of caspase 3 and cleaved-caspase 3 expression in three groups (Blank, Dox, EV) were suspended in 100 μl radio-immunoprecipitation assay (RIPA) buffer (Solarbio, Shanghai, China), the protein of which (30 μg) was subjected to 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Darmstadt, Germany). After being immersed in 5% nonfat milk for 2 h, they were incubated with primary antibodies overnight at 4 °C and secondary antibodies for 2 h at room temperature. The signal was detected by the Pierce enhanced chemiluminescence western blotting substrate (Millipore). The primary antibodies used for western blotting analysis were as follows: rabbit anti-Alix (Wanleibio, Shengyang, China), rabbit anti-CD9 (Abcam, Cambridge, UK), rabbit anti-CD63 (Wanleibio, Shengyang, China), rabbit anti-TSG101 (Abcam, Cambridge, UK), rabbit anti-caspase 3 (Wanleibio, Shengyang, China), rabbit anti-cleaved-caspase 3 (Wanleibio, Shengyang, China), and mouse anti-tubulin (Abcam, Cambridge, UK). Relevant experimental operations were following the manufacturer’s instructions.
5
Cellular Protein Detection Antibodies
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For cellular protein detection, the following antibodies were used: mouse anti-p53 (Abcam), mouse p21 (Santa Cruz, Dallas, TX, USA), rabbit anti-SLC31A1/CTR1 (Abcam), mouse anti-tubulin (Abcam) and mouse anti-GAPDH (Santa Cruz).
6
Immunoblot Analysis of Signaling Proteins
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Equal amounts of total protein extracts were separated by SDS-PAGE and electrotransferred to nitrocellulose membrane (GE-Healthcare Europe, Milan, Italy). Membranes were probed with rabbit anti-NLRP3 (Abcam, Cambridge, UK), rabbit anti-caspase-1 (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-Ser473Akt (Cell Signaling Technology, Danver, MA, USA), rabbit anti-total Akt (Cell Signaling Technology, Danver, MA, USA), rabbit anti-Ser9 GSK-3β (Abcam, Cambridge, UK), anti-total GSK-3β (Cell Signaling Technology, Danver, MA, USA), anti-Ser660 PKC and total PKC (Santa Cruz Biotechnology, Dallas, TX, USA), SOD2 (Novus Biologicals, Centennial, CO, USA), and NRF2 (Thermo Fisher Scientific, Whaltam, MA, USA) followed by incubation with appropriate HRP-conjugated secondary antibodies (BioRad). Proteins were detected with Clarity Western ECL substrate (BioRad, California, USA) and quantified by densitometry using analytic software (Quantity-One, BIO-RAD Image Lab Software.6.0.1.). Results were normalized with respect to densitometric value of mouse anti-tubulin (Abcam, Cambridge, UK), and autoradiograms showing statistically significant differences in terms of gel-loading homogeneity were excluded from the following biomarkers analyses.
7
Immunofluorescence Labeling of Oocytes
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Oocytes were incubated with 0.5% Triton X-100 for 5 min and then fixed with 4% paraformaldehyde for 30 min. Fixed oocytes were blocked in PBS containing 1% BSA, 0.1% Tween-20 and 0.01% Triton X-100 for 1 h. Oocytes were incubated for 48 h at 4 °C with primary antibodies: rabbit anti-CENPF (Abcam ab5; 1:700), mouse anti-Lamin B1 (Abcam ab8982; 1:400), human anti-centromere (Antibodies Incorporated 15-234-0001; 1:500), mouse anti-DRP1 (Abcam ab56788; 1:200), mouse anti-Tubulin (Abcam; 1:1000) rabbit anti-Tubulin (Abcam ab6046; 1:500) and mouse anti-cMyc (Thermo Fisher R950-25; 1:200). Secondary antibodies used were Rhodamine (TRITC) AffiniPure Donkey Anti-Rabbit (Jackson 711-025-152; 1:750), Alexa Fluor 647 AffiniPure Donkey Anti-Human (Jackson 709-605-149; 1:500), and Alexa Fluor 488 AffiniPure Donkey Anti-Mouse (Jackson 715-545-151; 1:500). DNA was stained with 10 μg/ml Hoechst 33342 (Sigma-Aldrich, St. Louis, MO, USA).
8
Antibodies and Dilutions for Protein Analysis
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The antibodies and their dilutions used in this study are as follows: mouse anti-Abd-B [Developmental Studies Hybridoma Bank (DSHB), 1A2E9] [immunofluorescence (IF) 1:40], rabbit anti-Pc [IF 1:20, Western blotting (WB) 1:4,000] (gift from S. Hirose), mouse anti-Myc (Santa Cruz, 9E10) (IF 1:50, WB 1:1,000), mouse anti-FLAG M2 (Sigma Aldrich, F1804) (ChIP 5 μl, WB 1:1,000), mouse anti-Tubulin (Abcam, ab7291) (WB 1:2,000), mouse anti-RNA pol-II (Abcam, ab5408) (ChIP 2 μl, IF 1:100), mouse anti-Lamin (DSHB, ADL67.10-s) (WB 1:1,000), rabbit anti-CCT7 (Atlas antibodies, HPA008425) (ChIP 8 μl). Secondary antibodies used for WB were goat anti-rabbit immunoglobulin G (IgG) H&L [horseradish peroxidase (HRP)] (Abcam, ab6721) and goat anti-mouse IgG H&L (HRP) (Abcam, ab6789) at 1:5,000 dilution. Secondary antibodies for IF were Cy3-conjugated goat anti-rabbit IgG (H+L) (Thermo Fisher Scientific, A10520) and Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) (Thermo Fisher Scientific, A28175) at 1:100 dilution.
9
Western Blot Analysis of EMT Markers
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Cells were lysed in western and IP cell lysis buffer (GenStar BioSolutions) that was supplemented with phenylmethylsulfonyl fluoride (Roche Diagnostics) for 10 min at 4 °C, and then centrifuged at 12,000 ×g for 10 min at 4 °C. The total protein of the supernatants was measured with the Bicinchoninic Acid Protein Assay Kit (GenStar BioSolutions). The protein extracts were electrophoresed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (GE Healthcare Life Sciences). After blocking, the membranes were incubated at 4 °C overnight with the following primary antibodies: Mouse anti-TPM3 (1:1,000; Abcam), rabbit anti-MMP2 (1:1,000; Abcam), rabbit anti-MMP9 (1:1,000; Abcam), mouse anti-tubulin (1:5,000; Abcam), mouse anti-β-actin (1:1,000; Abcam), and an Epithelial-Mesenchymal Transition (EMT) Antibody Sampler Kit (Cell Signaling Technology, Inc.). After 3 washes of 10 min each in tris-buffered saline and polysorbate (TBST; cat. No. 54124; Thermo Fisher Scientific, Inc.), the membranes were incubated with secondary antibodies (cat. No. 78543; Abcam, Inc.) at 1:2,000 dilution for 1 h at 25 °C, and an enhanced chemiluminescence reagent (GenStar BioSolutions) was used to develop the blots.
10
Western Blot Analysis of Protein Markers
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Cells were lysed and the protein concentration was quantified using the Pierce BCA Protein Assay kit (Thermo Scientific, Rockford, IL, EUA). Proteins were separated by electrophoresis on a SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (GE Healthcare, Waukesha, WI, USA). Membranes were blocked with 5% (w/v) milk powder in PBS for 1 h and incubated for 16 h at 4 °C with one of the following primary antibodies: rabbit anti-SLC31A1/CTR1 (Abcam, Cambridge, UK), mouse anti-GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-XPF (Thermo Fisher, Waltham, MA, USA), mouse anti-ERCC1 (Santa Cruz Biotechnology), mouse anti-Tubulin (Abcam), rabbit anti-NRF2 (Abcam). Afterwards, the membranes were incubated with the correspondent secondary antibody and a chemiluminescent HRP substrate (Merck Millipore. Burlington, MA, USA) was used to develop the membranes. Each blot was performed twice, and quantification of protein levels was done with the ImageJ software37 (link).
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