Protein analysis was performed as described previously [29 (link)] on whole hippocampal samples of all treatment groups. Briefly, 20μg of protein were resolved in AnyKD™ precast polyacrylamide gel (Bio-Rad Laboratories Inc., Hercules, CA, USA) and transferred to nitrocellulose membranes. Membranes were incubated with LI-COR blocking buffer (LI-COR, Lincoln, NE, USA) for 1h at room temperature before reacting overnight at 4°C with primary antibodies: Neuregulin-3 (NRG3) (1:500, PA5–18552, Invitrogen, Carlsbad, CA), ErbB4 (1:500, NBPI-33120, Novus, Centennial, CO.), and Beta-tubulin (1:2000, 2128L, Cell Signaling Technology, Danvers, MA.). After washing in phosphate buffered saline-Tween-20, the blots were incubated in fluorescent secondary antibodies (1:20000, LI-COR) in LI-COR blocking buffer for 1 h at room temperature. Membranes were then washed, and immunolabeling detection and densitometry measurements were performed using the LICOR Odyssey System (LI-COR). Ratios of the proteins of interest (NRG3 and ErbB4) to the housekeeping protein (β-actin) densities were calculated for each sample and normalized to 4-week old saline-treated controls.
Any kd precast polyacrylamide gel
Manufactured by Bio-Rad
Sourced in United States
The Any kD precast polyacrylamide gel is a laboratory equipment product designed for protein electrophoresis. It features a uniform gel matrix that can separate proteins based on their molecular weight, without the need for manual casting. The gel is pre-made and ready-to-use, providing a convenient and consistent solution for protein analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Blocking buffer, by LI COR (1 mentions)
Odyssey system, by LI COR (1 mentions)
β tubulin, by Cell Signaling Technology (1 mentions)
Gel doc imaging system, by Bio-Rad (1 mentions)
Coomassie blue reagent, by Thermo Fisher Scientific (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Skim milk, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail set 3, by Merck Group (1 mentions)
Anti foxo3 antibody, by Cell Signaling Technology (1 mentions)
Anti phospho akt thr308 antibody, by Cell Signaling Technology (1 mentions)
Anti p53 antibody, by Merck Group (1 mentions)
Anti akt pan antibody, by Cell Signaling Technology (1 mentions)
Anti phospho akt ser473 antibody, by Cell Signaling Technology (1 mentions)
Ip lysis buffer, by Thermo Fisher Scientific (1 mentions)
γ 32p atp, by GE Healthcare (1 mentions)
Foxo3, by Abnova (1 mentions)
Odyssey fc dual mode imaging system, by LI COR (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
6 protocols using any kd precast polyacrylamide gel
1
Quantitative Western Blot Analysis of Hippocampal Proteins
2
Enzymatic Modulation of α-Synuclein Aggregation
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Recombinant α-synuclein (rαSyn; 200ug/mL; Anaspec, USA) was added to Kgp or RgpB (a kind gift of Barbara Potempa, University of Louisville) to 100 mM Tris, 75 mM NaCl, 2.5 mM CaCl2, 10 mM Cys-HCl pH 7.5 buffer at 37 °C. The reaction was stopped at the indicated times (0.5, 1, 2, 5 min) by adding 4x Laemmli buffer (BioRad, USA) with 10% 2-mercaptoethanol. Samples were heated to 95 °C for 10 min and loaded on an Any kD precast polyacrylamide gel (BioRad, USA). After gel electrophoresis gels immersed in Coomassie Blue Reagent (ThermoFisher, USA) for 1 h at room temperature followed by imaging using a BioRad GelDoc imaging system.
3
Western Blot Analysis of Protein Expression
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Cells were lysed on ice with IP lysis buffer (Thermo Scientific, Waltham, MA) containing protease inhibitor cocktail set III (Millipore, Billerica, MA). Total proteins were separated by electrophoresis using Any kD precast polyacrylamide gel (Bio-Rad, Hercules, CA), and transferred onto PVDF membrane. After blocking with 5% skim milk (Thermo Scientific) in TBST buffer, membranes were incubated with the first antibody, respectively: anti-MELK monoclonal antibody (in-house, previously described [8 (link)]), anti-β-actin antibody, anti-p21 antibody, anti-FOXO1 antibody, anti-FOXO3 antibody, anti-pan-AKT antibody, anti-phospho-AKT (Thr308) antibody, anti-phospho-AKT (Ser473) antibody (Cell Signaling, Danvers, MA), and anti-p53 antibody (Sigma-Aldrich). β-actin was used as a loading control.
4
MELK Kinase Phosphorylation Assay
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MELK recombinant protein was kindly obtained from OncoTherapy Science Inc. As a control for the in vitro kinase assay, Histone H3 recombinant protein (EMD Millipore) was used as a positive control. In each reaction, 0.15 μM of FOXO1 (EMD Millipore), FOXO3 (Abnova, Taipei, Taiwan), or Histone H3 recombinant protein was mixed with 0.15 μM of MELK recombinant protein in 50 μl of kinase buffer and incubated for 2 hours at 30 °C. The kinase buffer contained 50 mM Tris-HCl, 10 mM NaCl, 10 mM MgCl2, 10 mM NaF, 1 mM Na3VO4, 1 mM DTT, 0.1 mM EDTA with 50 μM cold-ATP and 10 mCi of [γ-32P]ATP (GE Healthcare). The reaction was terminated by addition of SDS sample buffer and boiled for 5 min. Finally, the reacted samples were electrophoresed on Any kD precast polyacrylamide gel (Bio-Rad), transferred onto the PVDF membrane, and then autoradiographed with X-ray films.
5
Protein Extraction and Western Blot Analysis
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Protein extraction was performed in Pierce™ RIPA buffer (Thermo Fisher Scientific, 89901), supplemented with a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, 1861282), employing a handheld tissue ruptor. Protein concentration was quantified using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, 23225). An equal amount of proteins were run in Any KD precast polyacrylamide gel (Bio-Rad Laboratories) and transferred onto polyvinylidene fluoride (PVDF) membrane. Primary antibodies used were as follows: FHL2 (Medical and Biological laboratory, K0055-3, 1:1000) for mouse muscle lysate, FHL2 (Atlas Antibodies, HPA005922, 1:1000) for human muscle lysate, and GAPDH (Abcam, AB8245, 1:2000). Anti-rabbit IgG, HRP-linked secondary antibody (Cell signaling, 7074, 1:2000) and anti-mouse IgG HRP-linked secondary antibody (Cell signaling, 7076, 1:2000) were used before membrane underwent incubation with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, 34580) and analyzed using an Odyssey Fc Dual-Mode Imaging System (LI-COR Biotechnology). Uncropped blots, including all samples, are displayed in the Source data file for Fig. 3 .
6
Western Blot Analysis of Neural Markers
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Denatured total protein extract from the different samples was loaded on Any KD precast polyacrylamide gel (Bio-Rad) and transferred to nitrocellulose membranes using Trans-Blot Turbo Mini Nitrocellulose ready-to-use transfer Packs (Bio-Rad). Blots were incubated overnight at 4°C with primary antibodies dilution, for 2 hours at room temperature with HRPconjugated secondary antibodies (1:2000), (Figure S2 (Supporting Information)). Detection was carried out using ECL™ Western Blotting Reagents (GE Life Sciences) and signal detection and acquisition was carried out in an Amersham Imager 680 blot gel imager [AI680] (GE Life Sciences). The primary antibodies used are as follows: 1) Rabbit Anti-Tyrosine Hydroxylase Antibody (TH), 1:1000 (Pel-Freez #P40101-0), 2) Rabbit Anti-Glial Fibrillary Acidic Protein (GFAP), 1:2000 (Dako #Z0334), 3) Goat Anti-Doublecortin antibody (C-18) (DCX), 1:1000 (Santa Cruz #sc-8066), 4) Mouse monoclonal Anti-β-Actin antibody (ACTB), 1:1000 (Sigma #A5441). For western blot quantitation, data from bands and their background acquisition was performed using ImageJ software. Blots were normalized to the loading control, and final results were calculating using TCP as control sample. Statistical t-test analysis was carried out with GraphPad Prism statistical software (n=3 biological replicates).
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