Readyprep 2d starter kit

Manufactured by Bio-Rad

Sourced in United States

The ReadyPrep 2D Starter Kit is a laboratory equipment product designed for two-dimensional (2D) gel electrophoresis. It provides the necessary components and reagents to perform the initial steps in the 2D gel electrophoresis process.

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9 protocols using readyprep 2d starter kit

Comprehensive Protein Characterization by 2D-PAGE

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The two-dimensional (2D)-PAGE method was used to
separate the proteins based on their isoelectric point and their molecular
weight. This characterization allows further understanding of the
degradation of the proteins in this mix. A ReadyPrep 2-D Starter kit
(Bio-Rad, Hercules, CA) was used following the instruction manual.
The process has three main parts: sample preparation, isoelectric
focusing, and separation of proteins according to their molecular
weight by SDS-PAGE.

Mayta-Apaza A.C., Rocha-Mendoza D., García-Cano I, & Jiménez-Flores R. (2022). Characterization and Evaluation of Proteolysis Products during the Fermentation of Acid Whey and Fish Waste and Potential Applications. ACS Food Science & Technology, 2(9), 1442-1452.

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Comparative Venomics via 2D SDS-PAGE

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We performed two dimensional (2D) SDS-PAGE gel electrophoresis experiments using each of the four A. contortrix venoms to compare venom compositional profiles. For each gel, 0.5 mg of venom was prepared for 2D gel electrophoresis using the ReadyPrep™ 2-D Cleanup Kit for isoelectric focusing (IEF) (Bio-Rad) as per the manufacturer’s instructions. Cleaned-up venom samples were then applied to 7 cm, pH 3–10, non-linear IPG strips (Bio-Rad) using the ReadyPrep™ 2-D starter kit (BioRad), as per manufacturer’s instructions, and re-hydrated overnight at room temperature. After re-hydration, IEF was performed using a PROTEAN^® IEF Cell (Bio-Rad) with the manufacturer’s standard electrophoresis protocol for 7 cm IPG strips (default cell temperature = 20 °C; maximum current 50 Ua/strip; voltage = 250 V with linear ramp for 20 min; 4000 V with linear ramp for 2 hours; 4000 V with rapid ramp for 10,000 V-hr). After IEF, IPG strips were equilibrated (as per the ReadyPrep™ 2-D starter kit) and loaded onto Mini-PROTEAN TGX AnyKd precast gels (Bio-Rad) and run at 200 V for 35 minutes. Gels were then rinsed in water and stained with G-250 coomassie blue stain (Bio-Rad) for 1 hr to visualise proteins. Original (unedited) gel images can be found in Fig. S2.

Calvete J.J., Casewell N.R., Hernández-Guzmán U., Quesada-Bernat S., Sanz L., Rokyta D.R., Storey D., Albulescu L.O., Wüster W., Smith C.F., Schuett G.W, & Booth W. (2018). Venom Complexity in a Pitviper Produced by Facultative Parthenogenesis. Scientific Reports, 8, 11539.

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Quantification of CwlM Phosphorylation by 2D Gels

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For 2D gels and phosphothreonine quantification of CwlM, CwlM-FLAG was immunoprecipitated by bead beating cells in 50 mM Tris pH7.5, 300 mM NaCl with Protease Inhibitor Cocktail (Roche, Switzerland). α-FLAG M2 Magnetic Beads (Sigma Aldrich, Natick, MA) were added to supernatants and washed with 50 mM NaHPO_4, 300 mM NaCl. CwlM was eluted from the beads with 0.5 mg/ml FLAG peptide in TBS. Samples were separated in 2D using the ReadyPrep 2D Starter Kit (BioRad, Hercules, CA), according to the manufacturer’s protocols. SDS-PAGE was done with 4–12% NuPAGE Bis Tris precast gels (Life Technologies, Beverley, MA). Mouse α-FLAG (Sigma Aldrich) was used at 1:10,000 in TTBS, Rabbit α-strep (Genscript, China) and Rabbit α-phosphothreonine (Cell Signaling Technology, Danvers, MA) were used at 1:1000 in TTBS + 0.5% BSA.

Boutte C.C., Baer C.E., Papavinasasundaram K., Liu W., Chase M.R., Meniche X., Fortune S.M., Sassetti C.M., Ioerger T.R, & Rubin E.J. (2016). A cytoplasmic peptidoglycan amidase homologue controls mycobacterial cell wall synthesis. eLife, 5, e14590.

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2-DGE Procedure on Bio-Rad PROTEAN IEF

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2-DGE was performed on Bio-Rad PROTEAN IEF cell. The ReadyPrep 2-D Starter Kit, ReadyStrip IPG Strips, Ready Gel precast gel, mineral oil, 10X Tris/glycine/SDS Buffer, and paper wicks were purchased from Bio-Rad (USA). Buffers were prepared according to the kit protocol. 125 µL of freshly prepared rehydration/sample buffer for 7 cm IPG strip was added in a conical centrifuge tube containing the sample pellet, and vortexed to dissolve the pellet. The sample was centrifuged to settle down the fine particles. The whole procedure was performed according to the kit protocol. Gel images were taken through Gel DOC 800 system (Bio-Rad, USA).

Raza S.K., Saleem M., Shamsi T., Choudhary M.I., A.t.t.a.-.u.r.-.R.a.h.m.a.n, & Musharraf S.G. (2017). 5D proteomic approach for the biomarker search in plasma: Acute myeloid leukaemia as a case study. Scientific Reports, 7, 16440.

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Proteomic analysis of milk proteins

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Samples of 250 μg of milk protein solubilized in a rehydration buffer provided in the ReadyPrep 2-D Starter Kit (Bio-Rad Laboratories, Hercules, CA, USA) were loaded onto pH gradient 11 cm IPG gel strips (pH 4–7 for casein proteins and pH 3–10 for β-Lg; Bio-Rad Laboratories, Hercules, CA, USA), by passive rehydration for 4 h and active rehydration for 10 h at 50 V. Isoelectric focusing of the strips was performed using the Protean IEF Cell (Bio-Rad Laboratories, Hercules, CA, USA) at 20 °C using a 3 step protocol (250 V, 20 min and linear ramp; 8,000 V, 2.5 h, linear ramp and 8,000 V; 2.5 h, 20–30 kV-h, rapid ramp). The strips were equilibrated for 10 min each in equilibration buffer I and II provided in the ReadyPrep 2-D Starter Kit and finally in Tris-glycine-SDS running buffer. The equilibrated strip and the molecular weight marker Precision Plus Protein Standard Plug were added to the top of a Criterion TGX Stain-Free Precast IPG + 1Well Gel, overlaid with agarose and resolved in two dimensions. After 2-DE gel imaging, the gels were further subjected to western blot analysis, performed in the same conditions described in the previous section.

Geicu O.I., Stanca L., Dinischiotu A, & Serban A.I. (2018). Proteomic and immunochemical approaches to understanding the glycation behaviour of the casein and β-lactoglobulin fractions of flavoured drinks under UHT processing conditions. Scientific Reports, 8, 12869.

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Protein Purity Analysis by SDS-PAGE

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Extracts quality, protein purity and apparent molecular mass were monitored by SDS-PAGE using Mini-Protean II (Bio-Rad) systems with separating 17% polyacrylamide gels. Coomassie Brilliant Blue R-250 (Merk, Darmstadt, Germany) was employed for staining (CBS). Precision Plus Protein TM All Blue (Bio-Rad, Hercules, CA, USA) molecular mass leaders were used as reference.
Two-dimensional-electrophoresis (2D SDS-PAGE) was performed with purified proteins (10 µg) to analyze their isoelectric points (pI) and further detection by CBS, employing for the first dimension the ReadyPrep™ 2D starter kit (Bio-Rad) and IPG strips (Bio-Rad, Hercules, CA, USA) pH 3–10 gradient. The second dimension was carried out in an SDS-PAGE as described above.

Bueno-Díaz C., Martín-Pedraza L., Parrón J., Cuesta-Herranz J., Cabanillas B., Pastor-Vargas C., Batanero E, & Villalba M. (2021). Characterization of Relevant Biomarkers for the Diagnosis of Food Allergies: An Overview of the 2S Albumin Family. Foods, 10(6), 1235.

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Quantitative Analysis of PARK7 Protein

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The lysates of cells (150 μg) were resuspended in a rehydration buffer (Bio-Rad, ReadyPrep™ 2-D Starter Kit) at a final concentration of 2 to 4 mg/ml. Samples were separated using an isoelectric-focusing phoresis gel (pH 5 to 8). The first-dimension gels were subsequently separated on 10% Tris-HCl SDS-PAGE gels, and the levels of PARK7 were detected using western blotting. 2D SDS-PAGE standards (Bio-Rad, 1610320) were used to calibrate pI and molecular weights.

Wang B., Cai Z., Tao K., Zeng W., Lu F., Yang R., Feng D., Gao G, & Yang Q. (2016). Essential control of mitochondrial morphology and function by chaperone-mediated autophagy through degradation of PARK7. Autophagy, 12(8), 1215-1228.

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2D-PAGE Protein Separation Protocol

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2D-PAGE was performed using ReadyPrep 2D Starter Kit, ReadyStrip IPG strips 7 cm, pH 3-10 nonlinear and 4-20% Mini-Protean TGX gel (all from Bio-Rad, Hercules, CA, USA). Samples were dialyzed against deionized water using Slide A Lyzer Dialysis Cassettes (Thermo Fisher Scientific, Waltham, MA, USA) with 2 kDa cut-off and lyophilized.
Purified samples were dissolved in 300 μl of rehydration/ sample buffer and the IPG strip was passively rehydrated using 125 μl of reconstituted sample overnight at room temperature. Then, IEF was running with the maximum current of 50 μA/ strip. After IEF, IPG strips were equilibrated according to the manufacturer's instructions and placed on the top of the Mini-Protean TGX gel. Gel electrophoresis was performed at constant voltage of 160 V. Approximately, 4 µg of each samples was loaded on 2D SDS-PAGE. The protein visualization was carried out using Coomassie Brilliant Blue G 250 dye.

Strouhalova D., Toporova L., Lastovickova M., Macejova D., Bobalova J, & Brtko J. (2019). Novel insights into the combined effect of triorganotin compounds and all-trans retinoic acid on expression of selected proteins associated with tumor progression in breast cancer cell line MDA-MB-231: Proteomic approach. General physiology and biophysics, 38(2).

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Two-Dimensional Protein Separation Protocol

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Two-dimensional separation of tested protein isolates was run using a commercial Ready Prep 2D Starter Kit (Bio Rad) following the manufacturer's protocol. Upon completion of the separation, the gels were stained overnight with a staining buffer. They were destained with a destaining buffer for 3 h. The gels were documented using a gel documentation system and analyzed with the PDQuest 2D Gel Analysis software (Bio-Rad).

Konieczna C., Olejnik-Schmidt A, & Schmidt M.T. (2018). Lactobacillus spp. belonging to the Casei group display a variety of adhesins. Acta scientiarum polonorum. Technologia alimentaria, 17(1).

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