Protein samples were loaded into a 10% Bis–Tris NuPAGE gel and electrophoresed in SDS-MES buffer alongside a protein molecular weight standard; EZ-Run™ Prestained Rec Protein Ladder (Fisher BioReagents) or SuperSignal molecular weight protein ladder (Thermo). For protein concentration determination, samples of unknown concentration were loaded at multiple dilutions and electrophoresed alongside a V565 reference standard curve. The gels were imaged in an Imagequant LAS 4000 (Cytiva) using the white transmitted light table and analysed using Imagequant TL software to determine unknown protein concentrations from the V565 standard curve.
Ez run prestained rec protein ladder
Manufactured by Thermo Fisher Scientific
Sourced in United States
The EZ-Run™ Prestained Rec Protein Ladder is a pre-stained molecular weight ladder used for estimating the molecular weights of proteins in SDS-PAGE. It contains a mixture of recombinant proteins with precisely defined molecular weights and is pre-stained with a tracking dye for easy visualization.
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Lab products found in correlation
Supersignal molecular weight protein ladder, by Thermo Fisher Scientific (1 mentions)
Imagequant las 4000, by Cytiva (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Zncl2, by Merck Group (1 mentions)
Cacl2, by Merck Group (1 mentions)
Amersham hybond ecl, by GE Healthcare (1 mentions)
G1544, by Merck Group (1 mentions)
As05 092, by Agrisera (1 mentions)
Mini protean 3 cell, by Bio-Rad (1 mentions)
Kd mini protean tgx precast gel, by Bio-Rad (1 mentions)
Mini protean electrophoresis cell, by Bio-Rad (1 mentions)
Gelcode blue safe protein stain, by Thermo Fisher Scientific (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
8 protocols using ez run prestained rec protein ladder
1
SDS-PAGE Protocol for Protein Quantification
2
Nitrilase Protein Expression and Detection
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Overnight cultures were diluted 1:100 in 3 ml LB kanamycin and grown to an OD600 of 0.3 at 37°C, at which point 1 mM IPTG was added for 3–4 h and induction continued at 30°C. Cells from 3 ml of induced culture were pelleted by centrifugation and washed twice with 0.1 M MOPS pH 7.7. The cell pellet was resuspended in 300 μl of 0.1 M MOPS pH 7.7 and sonicated on ice with 10 bursts of 10 s each using a microprobe. Cells debris was centrifuged and the supernatant was recovered and transferred to a new tube. The cell debris was also resuspended in 300 μl of 0.1 M MOPS pH 7.7. 10 μl of the supernatant or the resuspended cell debris suspension were mixed with 3 μl of Amresco 4X Next Gel Sample Loading Buffer, boiled for 10 min and run on an Amresco Pro-Pur Next Gel 10% polyacrylamide gel along with Ez-Run Prestained Rec Protein Ladder (Fisher Bioreagents). The gel was then electroblotted to a nitrocellulose membrane (Whatman) and anti-nitrilase polyclonal rabbit serum was used as primary antibody (1:3000) and HRP conjugated goat anti-rabbit IgG as the secondary antibody. SuperSignal West Pico rabbit IgG Chemiluminescent detection kit (Thermoscientific) was used to visualize the protein bands.
3
Quantifying Gelatinase Activity in Senescent Fibroblasts
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Fresh conditioned media was collected from fibroblasts and concentrated using vivaspin-500 5 kDa cut-off spin columns (Sartorius, #VS0101) by centrifugation at 10,000 rpm for 15 m. Concentrated conditioned media from senescent and presenescent fibroblasts were mixed with 2X non-reducing zymography sample loading buffer (Tris-HCl, 200 mM; SDS, 2% (w/v); glycerol, 20% (v/v) and bromophenol blue 0.1% (w/v), pH 6.8), and incubated for 30 min at 37oC. Samples were electrophoresed in polyacrylamide gels containing gelatin (10% (w/v)). Following electrophoresis gels were allowed to renature in 2.5% (v/v) Triton X-100 in PBS for 1 h and maintained in zymogram developing buffer; 0.5 M Tris, 2 M NaCl (BDH GPR), 50 mM CaCl2 (Sigma), 50 μM ZnCl2 (Sigma), 1% (v/v) Triton X-100, pH=7.5; at 37°C for 24 h with gentle agitation. Gels were then stained with Coomasie brilliant blue R-250 for 30 m and destained with 40% (v/v) methanol and 10% (v/v) glacial acetic acid until distinct faint bands became visible. Fisher Scientific's EZ-Run pre- stained Rec protein ladder was used as molecular weight markers. Gelatinase activity was determined using Quantity One 1D gel software (BioRad; version 4.5.0) and the intensity of bands were divided by the total number of fibroblasts present in each sample of conditioned medium to normalize to cell density.
4
SDS-PAGE Analysis of BSA-Capsaicin Nanoparticles
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Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using the Tris–glycine buffer system of Laemmli43 (link) with modifications. In addition, to estimate the protein molecular weight (MW) we used the EZ-Run pre-stained Rec protein ladder with bands in the range of 10–170 kDa (Fisher Scientific, Waltham, MA, USA). To reference the MW of native BSA, lyophilized BSA (66 kDa), was dissolved in deionized water to stock concentration [500 µg mL−1] (Equitech-Bio, Kerrville, TX, USA). From stock, the solution was adjusted to 20 µg per lane. Finally, the BSA-capsaicin nanoparticles after drying procedures were dissolved and adjusted at a concentration of 20 µg per lane. The samples were resolved on 12% SDS-PAGE at a constant voltage of 70 V for stacking and 80 V for resolving. Then, gels were washed three times with deionized water for 5 min each and were boiled for 1 min with CBB staining solution (0.025% Coomassie dye, G-250 in 10% acetic acid). Subsequently, gels were washed again with deionized water for 5 min and clarified to visualize polypeptide bands. Gels were captured (Documentation Systems Bio-Rad) at 600 dpi resolution in tagged image file (.tif) format with 1580 × 1489 pixels in grey scale. In this format, 0 was assigned to black and 255 to white in the grey scale.
5
Chloroplast Isolation and Fractionation
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Chloroplasts from rosette leaves collected 16 das were isolated as described by Grabsztunowicz and Jackowski (2012) , omitting the Percoll step gradient. Stroma and thylakoid membranes from isolated chloroplasts were separated as previously reported (Armbruster et al., 2010 (link)). A volume of isolated chloroplasts and thylakoids equivalent to 20 μg of chlorophyll, as well as 20 μg of proteins from the stroma fraction, including a lane with an EZ-Run Pre-stained Rec Protein Ladder (Fisher BioReagents), were resolved by 10% SDS–PAGE, blotted on nitrocellulose membranes (Amersham Hybond ECL, RPN203D; GE Healthcare), and subjected to immunoblot analysis with specific antibodies (G1544, Sigma; AS03 037-10 and AS05 092, Agrisera AB). Isolation of thylakoids from rosettes collected 16 das and blue native PAGE were carried out as reported in Pérez-Pérez et al. (2013) (link).
6
Protein Profiling by SDS-PAGE
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The protein profiles of SC, WC, skim milk, WPPC, and the insoluble protein at pH 4.6 of these samples were determined by SDS-PAGE performed on a Mini-Protean 3 Cell (Bio-Rad Laboratories). Nonreducing and reducing SDS-PAGE analyses were done on a 12% Tris-HCl ready gel with 10 wells (Bio-Rad Laboratories). Before loading, samples were defatted and diluted with an appropriate amount of buffer to obtain a final protein concentration of 1 μg/μL. For the reducing conditions, 5% of 2-mercapthoethanol was added. The mixture was then boiled for 5 min. Ten microliters of each sample (corresponding to 10 μg of protein) was loaded into each well. Electrophoresis was run for 35 min at 200 V in 0.025 M Tris-HCl buffer solution (pH 8.3, including 0.192 M glycine and 0.1% SDS, wt/vol) at room temperature. After the electrophoresis run was completed, the gel was stained with Coomassie Blue G-250. Gels were destained by immersing in deionized water. Apparent molecular masses were estimated by comparison with standard markers (EZ-RUN Prestained Rec Protein Ladder, Fisher BioReagents) ranging from 11 to 170 kDa.
7
SDS-PAGE Analysis of Skin Tissue Proteins
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1D SDS-PAGE was performed to observe protein bands in the skin tissue sample. 100 mg of each sample were homogenized with 300 µl saline and were then subjected to cooling centrifugation (VS-18000 M, Korea, power 220 V/50 Hz) (12,303×g for 30 min at 4 °C). 100 µl from the supernatant proteins were mixed with 500 µl acetone and left overnight at -20 °C. Before measurement, samples were centrifuged at 12,303×g for 3 min at 4 °C. Proteins were diluted with sample buffer (10% SDS, 20% Glycerol, 0.2 M trispH6.8, 10 mM beta-mercapto-ethanol, and 0.05% bromophenol blue) at the ratio of 1:4 and were then heated for 5 min at 95 °C. Proteins were loaded into the well of kD™ Mini-PROTEAN® TGX™ Precast Gel (Bio-Rad, Hercules, CA), and were run with Mini-PROTEAN electrophoresis cells (Bio-Rad, Hercules, CA) at 80 V for 4 h. EZ-Run Prestained Rec Protein Ladder (Fisher Bioreagents, Pittsburgh, PA) containing proteins from 16 to 270 kDa was used as a protein marker. Gels were stained with staining solution (50% dH 2 O, 40% methanol, and 10% glacial acetic acid) [43] .
8
SDS-PAGE Analysis of Protein Samples
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2μg of each antibody was diluted in Laemmli Sample Buffer (Bio-Rad) for analysis under nonreducing conditions or Laemmli Sample Buffer containing 10% ß-mercaptoethanol for analysis under reducing conditions. Samples were heated either at 95°C for 5 min (reduced) or at 90°C for 2 min (non-reduced). Samples and EZ-Run™ Prestained Rec Protein Ladder (Fisher BioReagents) were subjected to either 10% polyacrylamide gel at 120 V (reduced samples) or 8% polyacrylamide gel at 100 V (non-reduced samples). Gels were stained using GelCode™ Blue Safe Protein Stain (Thermo Scientific), fixed for 30 min in buffer containing 40 % ethanol and 10% acetic acid, destained with distilled water and scanned using ChemiDocTM Touch Imaging System (Bio-Rad).
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