Benchmark protein ladder

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

The BenchMark protein ladder is a set of pre-stained protein standards used for estimating the molecular weights of proteins in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) experiments. The ladder contains a mixture of proteins with known molecular weights, which can be used as a reference to determine the approximate size of unknown protein samples.

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Lab products found in correlation

Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions) Dc assay, by Bio-Rad (2 mentions) Q5 hot start high fidelity dna polymerase, by Thermo Fisher Scientific (2 mentions) Tri reagent, by Merck Group (2 mentions) Nupage bis tris gel, by Thermo Fisher Scientific (2 mentions) Pageblue protein staining solution, by Thermo Fisher Scientific (2 mentions) Bis tris gel, by Thermo Fisher Scientific (2 mentions) Nupage lds loading buffer, by Thermo Fisher Scientific (2 mentions) Coomassie brilliant blue g 250, by Merck Group (1 mentions) Imagemaster 2d platinum 7, by GE Healthcare (1 mentions) Tetra cell, by Bio-Rad (1 mentions) Holo transferrin, by Merck Group (1 mentions) Bcip nbt color development substrate, by Promega (1 mentions) Ap conjugated rabbit anti mouse igg, by Santa Cruz Biotechnology (1 mentions) Transferrin alexa 633, by Thermo Fisher Scientific (1 mentions) Hoechst 33342, by Thermo Fisher Scientific (1 mentions) Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions) Dulbecco s modified eagle medium dmem, by Merck Group (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Horseradish peroxidase conjugated goat anti rabbit antibody, by Merck Group (1 mentions)

29 protocols using benchmark protein ladder

Proteomic analysis of Bidens pilosa

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For 1-DE gel electrophoresis, samples of total extract and acetonic fraction of B. pilosa were assessed by 12% polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) in non-reducing conditions (25 (link)), in parallel with molecular weight markers (BenchMarkTM Protein Ladder 6–200 kDa, Invitrogen, Karlsruhe, Germany). Gels were stained with Coomassie brilliant blue G-250® (Sigma-Aldrich, St. Louis, MO, USA). The molecular weight bands were estimated by linear regression analysis, based on the calculation of relative mobility (Rf), using KODAK 1D Image Analysis program (Eastman Kodak Co., Rochester, USA). Additionally, the band was submitted to MS/MS and identified in the National Center for Biotechnology Information (NCBI) database.
For 2-DE gel electrophoresis, 60 μg of acetonic fraction were diluted in ultrapure water and separated by isoelectric focusing (IEF) on 7-cm immobilized pH gradient strips (ReadyStripTM IPG Strip pH 3–10) overnight at room temperature, following the manufacturer instructions (GE, Healthcare, Uppsala, Sweden). After IEF, strips were equilibrated and running onto precast 12% polyacrylamide gels, being the staining spots analyzed by ImageMasterTM 2-D Platinum 7.0 (GE Healthcare, Amersham Pharmacia Biotech, United Kingdom) to be submitted to MS/MS and identified in the NCBI database.

Mota C.M., Santiago F.M., Cardoso M.D., Rostkowska C., de Oliveira T.C., Silva D.A., Pirovani C.P., Mineo T.W, & Mineo J.R. (2019). Acetonic Fraction of Bidens pilosa Enriched for Maturase K Is Able to Control Cerebral Parasite Burden in Mice Experimentally Infected With Toxoplasma gondii. Frontiers in Veterinary Science, 6, 55.

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Salmonella Outer Membrane Protein Analysis

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Salmonella OMP preparations were analyzed by SDS-PAGE using a gel casting system (Bio-Rad tetra cell) and 12.5% isocratic Laemmli gels. Approximately 75 g of protein were loaded in each lane. Gels were run at constant amperage (20mA) until the bromophenol blue tracking front had run off the end of the gel. Gels were stained with 0.1% Coomassie blue R-250 dye at room temperature for 30 minutes, and then distained overnight with 10% acetic acid in distilled water. The range of OMP molecular weights was estimated from a standard size marker (Benchmark TM protein ladder, Invitrogen (Chicago, USA)).

Ferrer-Navarro M., Ballesté-Delpierre C., Vila J, & Fàbrega A. (2016). Characterization of the outer membrane subproteome of the virulent strain Salmonella Typhimurium SL1344. Journal of proteomics, 146.

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Immunofluorescence Staining Protocol

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BCIP-NBT Color Development Substrate was purchased from Promega Corporation (Madison, WI, USA). Alkaline phophatase (AP)-conjugated rabbit anti-goat IgG and AP-conjugated rabbit anti-mouse IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Holo-transferrin, bovine serum albumin, Roswell Park Memorial Institute-1640 (RPMI-1640) medium, Dulbecco’s modified Eagle medium (DMEM) and anti-transferrin goat IgG were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bench Mark Protein Ladder, albumin-Alexa 488, transferrin-Alexa 633, Hoechst 33342, goat anti-mouse antibody coupled to AlexaFluor 488 or 594, and rabbit anti-goat antibody coupled to AlexaFluor 594 were purchased from Invitrogen-Life Technologies (Carlsbad, CA, USA). Prolong Gold antifade reagent was purchased from Molecular Probes-Life Technologies (Eugene, OR, USA). Fetal bovine serum (FBS) was purchased from Cultilab Ltda (Campinas, SP, Brazil). The anti-Ssp4 2C2 monoclonal antibody [30 (link)] was kindly provided by Dr. Renato Mortara (UNIFESP, Brazil).

Batista C.M., Kessler R.L., Eger I, & Soares M.J. (2015). Trypanosoma cruzi Intracellular Amastigotes Isolated by Nitrogen Decompression Are Capable of Endocytosis and Cargo Storage in Reservosomes. PLoS ONE, 10(6), e0130165.

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Western Blot Analysis of WzzB Protein

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Bacteria were grown and induced as described above, harvested by centrifugation, and resuspended in 1x sample buffer [38 (link)]. Protein samples were heated at 100°C for 5 min, except for the cross-linking samples, which were heated at 60°C for 5 min, before loading onto 12% or 15% SDS-polyacrylamide gels. The electrophoresed protein samples were transferred to a nitrocellulose sheet (Medos), which was further subjected to incubation with polyclonal anti-WzzB_SF antibodies [21 (link)] at 1/500 dilution. Detection of the proteins was performed with goat anti-rabbit horseradish-peroxidase-conjugated antibodies (KPL) and chemiluminescence reagent (Sigma). The BenchMark protein ladder (Invitrogen) was used as molecular-mass standards.

Chang C.W., Tran E.N., Ericsson D.J., Casey L.W., Lonhienne T., Benning F., Morona R, & Kobe B. (2015). Structural and Biochemical Analysis of a Single Amino-Acid Mutant of WzzBSF That Alters Lipopolysaccharide O-Antigen Chain Length in Shigella flexneri. PLoS ONE, 10(9), e0138266.

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Protein Extraction and Western Blot Analysis

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Cells were lysed in 95 °C pre-heated SDS lysis buffer (0.1 M Tris-HCl pH 7.5, 10% SDS, 1 mM Na₄VO₃ and Complete^TM protease inhibitor mix (Roche)). Protein concentrations were determined by DC assay (BioRad, Hercules, USA) and samples were mixed with NuPage LDS sample buffer (Invitrogen, #NP0007). Equal amounts of protein were separated by SDS-PAGE using Criterion^TM 10% tris gels (Bio-Rad, 18 wells, #567-1034 and 26 wells, #567-1035) and premade Tris/Glycine/SDS running buffer (BioRad, #161-0732), and BenchMark protein ladder (Invitrogen, #10747-012). Proteins were transferred by Trans-Blot Turbo transfer system (BioRad) to a PVDF membrane (BioRad, #170-4159), stained with Ponceau S and blocked in 5% nonfat dry milk in TRIS-buffered saline with Tween (TBS-T; 0.01 M Tris/HCL, 0.15 M NaCl, 0.1% Tween 20, pH 7.4) for 1 h at 37 °C. Membranes were incubated with primary antibodies diluted in 5% nonfat dry milk in TBS-T overnight at 4 °C and with horseradish peroxidase (HRP) conjugated secondary antibodies in 5% nonfat dry milk in TBS-T for 1 h at room temperature. Bands were visualized by chemiluminescent using enhanced chemiluminescent (ECL) substrate (Pierce, #32106). Densitometric analyses were carried out using ImageJ.

Olesen C.W., Vogensen J., Axholm I., Severin M., Schnipper J., Pedersen I.S., von Stemann J.H., Schrøder J.M., Christensen D.P, & Pedersen S.F. (2018). Trafficking, localization and degradation of the Na+,HCO3− co-transporter NBCn1 in kidney and breast epithelial cells. Scientific Reports, 8, 7435.

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HA-tagged Protein Expression Protocols

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The pMOTag4H vector was a gift from Dr. Thomas Seebeck (University of Bern, Bern, Switzerland). Monoclonal antibody L8C4 was a gift from Dr. Phillippe Bastin (Institute Pasteur, Paris). Wild type and M9 mutated Bacillus subtilis glmS ribozymes were gifts from Dr. Vasant Muralidharan (University of Georgia). Q5® Hot Start High-Fidelity DNA polymerase, BenchMark protein ladder, Alexa-conjugated secondary antibodies were purchased from Invitrogen (ThermoFisher Scientific, Waltham, MA). Purified HA.11 clone 16B12 monoclonal antibody against HA was purchased from Covance (Biolegend, San Diego, CA). The protein assay reagent, Magic Marker, Zeta-Probe GT Genomic Testing blotting and nitrocellulose membranes were from Bio-Rad Laboratories (Hercules, CA). AMAXA human T-cell Nucleofector kit was purchased from Lonza (Allendale, NJ). The primers were purchased from Integrated DNA Technologies (Coralville, IO). TRI® reagent and all other reagents of analytical grade were from Sigma-Aldrich (St. Louis, MO).

Cruz-Bustos T., Ramakrishnan S., Cordeiro C.D., Ahmed M.A, & Docampo R. (2018). A Riboswitch-based Inducible Gene Expression System for Trypanosoma brucei. The Journal of eukaryotic microbiology, 65(3), 412-421.

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SDS-PAGE Protein Separation and Visualization

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100 μg of protein were applied in a 15% acrylamide separating gel was used with stacking at 4% for SDS-PAGE on SE 600 Ruby Standard Dual Cooled Vertical Unit. The acrylamide gel was run at 40 mA for 15 min and then at 100 mA for 2 h (Electrophoresis Power Supply – EPS 601 - GE Life Sciences) [30 (link)] in SDS buffer (124 mM Tris; 960 mM glycine; 17.5 mM SDS). 10 μl of protein molecular weight standard BenchMark™ Protein Ladder (Invitrogen) were used. At the end of electrophoresis, the gels were visualized by staining with Coomassie brilliant blue (5% acetic acid; 20% methanol; 0.2% Comassie Brilliant Blue R-250) and then decolorized with 0.5% acetic acid and 20% methanol.

Malafaia C.B., Guerra M.L., Silva T.D., Paiva P.M., Souza E.B., Correia M.T, & Silva M.V. (2015). Selection of a protein solubilization method suitable for phytopathogenic bacteria: a proteomics approach. Proteome Science, 13, 5.

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Affinity Chromatography Purification of Chick Embryo Proteins

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A total of 400 chick embryo trunks were fractionated by affinity chromatography on agarose-bound-PNA (Vector Labs), following procedures used previously in the laboratory (Davies et al., 1990 (link)). Care was taken to elute the affinity column with 0.5M NaCl 1% CHAPS (w/v) and 100 mM Tris-HCl (pH7.5), followed by elution with 0.4M lactose/2% CHAPS (w/v) in PBS. Eluates (20 μL) were concentrated using StrataClean Resin (Agilent Technologies) (Bonn et al., 2014 (link); Otto et al., 2017 (link)). Protein bound to the resin was eluted using SDS reducing sample buffer with heating for five minutes at 95°C, followed by centrifugation (10000 g for 1 min). The supernatant containing the proteins was fractionated on slab gels (7.5% acrylamide separating gel; 5% stacking gel). Samples were examined under reducing conditions and electrophoresis was performed in 25 mM Tris (pH 8.3), 192 mM glycine, 0.1% SDS. Molecular weight markers (BenchMark Protein Ladder, Invitrogen) were also run. The gel was developed with MS-compatible silver stain using the protocol of Blum et al., 1987 (link). The band was excised in a laminar flow hood and submitted for mass spectrometry analysis (Alta Bioscience, UK).

Cook G.M., Sousa C., Schaeffer J., Wiles K., Jareonsettasin P., Kalyanasundaram A., Walder E., Casper C., Patel S., Chua P.W., Riboni-Verri G., Raza M., Swaddiwudhipong N., Hui A., Abdullah A., Wajed S, & Keynes R.J. (2020). Regulation of nerve growth and patterning by cell surface protein disulphide isomerase. eLife, 9, e54612.

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Characterization of Purified SMTI

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The homogeneity of SMTI was analyzed on a 12% resolving gel by SDS-PAGE following Laemmli’s method^{55 (link)}. The molecular weight of the purified SMTI was determined by comparing with the corresponding relative movement of pre stained benchmark protein ladder (Invitrogen).

S.a.m.i.k.s.h.a., Singh D., Kesavan A.K, & Sohal S.K. (2019). Exploration of anti-insect potential of trypsin inhibitor purified from seeds of Sapindus mukorossi against Bactrocera cucurbitae. Scientific Reports, 9, 17025.

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Comprehensive Protein Sample Preparation

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Acetonitrile, deoxycholic acid (DC), NaCl and ponceau S were from Sigma (Sigma-Aldrich, Steinheim, Germany), and bromophenol blue, Coomassie Bue Brilliant R-250 and FA from Fluka (Fluka Chemie, Buchs, Switzerland). Tween-20 was bought from Roche Diagnostics (Mannheim, Germany). DTE, glycerol and urea were purchased from MP Biomedicals (Illkirch, France), ammonium bicarbonate and iodoacetamide from ICN Biomedicals (Aurora, Ohio, United States), 0.9% NaCl from Baxter (Volketswil, Switzerland), 10x PBS (1x PBS eq. to 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄ and 1.8 mM KH₂PO₄) from Laboratorium Dr. G. Bichsel (Interlaken, Switzerland), EDTA from Merck (MSD Merck Sharp & Dohme, Luzern, Switzerland), Tris-HCl from BIO-RAD (Hercules, CA, United States), ethanol from Thommen-Furler AG (Rüti bei Büren, Switzerland), Top Block from Lubio Science (Luzern, Switzerland), benchMark Protein Ladder (prestained or not) from Invitrogen (Carlsbad, CA, United States), FITC mouse anti-Human CD47 antibody from BD Pharmingen (BD Biosciences, Franklin Lakes, NJ, United States), and Sequencing Grade Modified Trypsin (Trypsin) from Promega AG (Dübendorf, Switzerland). Deionized water (18.2 MΩ⋅cm) was prepared using a Purelab option Q-15 (Elga LabWater).

Prudent M., Delobel J., Hübner A., Benay C., Lion N, & Tissot J.D. (2018). Proteomics of Stored Red Blood Cell Membrane and Storage-Induced Microvesicles Reveals the Association of Flotillin-2 With Band 3 Complexes. Frontiers in Physiology, 9, 421.

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