Protean ief

Manufactured by Bio-Rad

Sourced in United States

The PROTEAN IEF is a laboratory equipment designed for isoelectric focusing (IEF), a technique used to separate and analyze proteins based on their isoelectric point. The device provides a controlled environment for the IEF process, allowing for the effective separation and analysis of protein samples.

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Protean i12TM IEF cell Protean IEF Cell BioRad Protean IEF Cell Protean IEF Cell focusing unit Protean IEF system PROTEAN IEF unit PROTEAN® i12TM IEF system PROTEAN IEF apparatus BioRad PROTEAN IEF Cell system PROTEAN i12 IEF system

18 protocols using protean ief

Protein Quantification and Rehydration Protocol

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The soluble
fraction was collected and quantified using Bradford assay protein
quantification. The concentration was adjusted to 250 μg and
completely dried using a speedvac (Concentrator plus, Vacufuge plus,
Eppendorf, Hamburg, Germany). The samples were then rehydrated and
distributed in the channels from the PROTEAN IEF (Bio-Rad; Hercules,
CA). Then, the respective immobilized pH gradient (IPG) of pH 3–10,
11 cm ReadyStrip IPG (Bio-Rad; Hercules, CA) strips were placed gel
down. Mineral oil was overlaid on each strip to prevent evaporation.
Finally, rehydration was carried out at 50 V for 12 h.

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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Two-Dimensional Gel Electrophoresis of Cell Lysates

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HepG2 and SNU449 cell lysates were prepared and lysed in the rehydration sample buffer (Bio-Rad, Hercules, CA, USA). After being vigorously vortexed for 90 min at room temperature (RT), the samples were next centrifuged at 16,000× g under 4 °C temperature for 20 min. The supernatant was collected, and the protein concentration was measured by Bradford Protein Assay kit (Bio-Rad, Hercules, CA, USA). In the first-dimensional (1DE) analysis, 125 μL of 150 μg protein was loaded onto the isoelectric focusing (IEF) strip (Non-linear, pH 3–10, 7 cm, Bio-Rad, Hercules, CA, USA). Then, 1DE was performed in the PROTEAN IEF (Bio-Rad, Hercules, CA, USA) with linearly increased voltage from 0 to 250 V in 30 min, then gradually increased voltage to 400 V in 90 min, and finally kept at 4000 V for 25,000 kvh. The prepared IEF strips were subsequently used for the second-dimensional (2DE) gel electrophoresis. Strips were docked into 12% SDS-polyacrylamide (SDS-PAGE) gel by Overlay agarose (Bio-Rad, Hercules, CA, USA), and the electrophoresis was accomplished under a constant voltage of 80 V for 2 h. The SDS-PAGE gels were then transferred onto the nitrocellulose (NC) membrane (Osmonics Inc., Westborough, MA, USA) or stained with 0.1% Coomassie blue R-250 reagent. The protein spots were identified by PDQuest 2-DE analysis software v. 8.0(Bio-Rad, Hercules, CA, USA).

Ma Y., Qiu C., Wang B., Zhang X., Wang X., Aguilera R.J, & Zhang J.Y. (2022). Autoantibody against Tumor-Associated Antigens as Diagnostic Biomarkers in Hispanic Patients with Hepatocellular Carcinoma. Cells, 11(20), 3227.

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Proteomic Analysis of Flagellar Proteins

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Two-dimensional (2D) gel electrophoresis was carried out as previously described (Kurniyati et al., 2017 (link)). Equal amounts of purified PFs were resuspended in a rehydration buffer (5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10, 0.2% Bio-Lyte 3/10 Ampholyte, 40 mM Tris, and 0.0002% Bromophenol Blue) and then subjected to separation using 7 cm long pH 5→8 linear IPG strips. The first dimension of isoelectric focusing (IEF) was performed using PROTEAN IEF (Bio-Rad Laboratories, Hercules, CA), followed by equilibration according to the manufacturer's protocol. The second-dimension separation was carried out using sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis (SDS-PAGE) as described previously (Kurniyati et al., 2017 (link)). The resultant gels were subjected to Coomassie blue staining or immunoblotting analyses. The antibodies against T. pallidum FlaBs and T. denticola FlaA and DnaK are described in our previous publications (Kurniyati et al., 2017 (link), Kurniyati & Li, 2016 (link)). The stoichiometry of each flagellar filament protein was analyzed using Image Lab software from Bio-Rad (Bio-Rad).

Kurniyati K., Chang Y., Liu J, & Li C. (2022). Transcriptional and functional characterizations of multiple flagellin genes in spirochetes. Molecular Microbiology, 118(3), 175-190.

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Detailed 2-DE Protein Separation Protocol

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2-DE was performed as previously described [13] (link) using the PROTEAN IEF and PROTEAN Plus Dodeca cell systems (BioRad). Total protein (300 µg/250 µL) pre-mixed with rehydration buffer was run in an IEF system using a precast 11 cm IPG strip with a pI range of 3–10 (BioRad). Passive rehydration was performed for 12 hours to introduce the protein samples onto the IEF strip. IEF was performed as follows: slow 250 V for 30 minutes, rapid 1,000 V for 30 minutes, linear 8,000 V for 4 hours, and rapid 8,000 V for 5 hours. Each run required approximately 24 hours for completion. The total Vh ranged from 60,000–69,000. Following IEF, the IPG strips were equilibrated in equilibration buffer I (6 M urea, 2% SDS, 0.375 M Tris-HCl, pH 8.8, 20% glycerol, and 2% DTT) for 15 minutes, then equilibrated in equilibration buffer II (DTT was replaced with 2.5% IAA) for 15 minutes. The continuous 10% linear gradient SDS-PAGE gels onto which the strips were loaded were electrophoresed at a constant voltage of 120 V for 2–3 hours until the bromophenol blue reached the bottoms of the gels. The gels were stained with “blue silver”, a very sensitive colloidal Coomassie G-250 staining dye compatible with MS analysis.

Li W., Wang W., Li Y., Wang W., Wang T., Li L., Han Z., Wang S., Ma D, & Wang H. (2014). Proteomics Analysis of Normal and Senescent NG108-15 Cells: GRP78 Plays a Negative Role in Cisplatin-Induced Senescence in the NG108-15 Cell Line. PLoS ONE, 9(3), e90114.

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Two-dimensional Gel Electrophoresis of Muscle Proteome

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The sarcoplasmic and myofibrillar proteome (800 μg, respectively) was included in the Bio-Rad buffer (8 M urea, 2% CHAPS (w/v), 50 mM DTT, 0.3% carrier ampholyte (v/v), bromophenol blue). The samples were loaded on the IPG strips (immobilized pH gradient, 24 cm, pH 3–10, Bio-Rad). The PROTEAN II XL and PROTEAN IEF (isoelectric focusing) cell system (Bio-Rad, Hercules, CA, USA) performed in the first and second dimension electrophoreses, respectively.
In the first dimension, IEF was subjected to passive rehydration (16 h) and then rapid voltage ramping (80,000 V h) was applied. In the second dimension, proteins were resolved on 12% SDS-PAGE gels, which were stained in Coomassie Brilliant Blue (48 h). Gel images were scanned by an image scanner (ImageScanner III, GE Healthcare, Branford, CT, USA). ImageMaster 2D Platinum software (6.0 version, GE Healthcare, Branford, CT, USA) was used to analyze the scanning images. Through expressing the relative number of each spot as the ratio of the number of single spots to the total number of valid spots, the detected and matched spots were normalized. The mean values of gels for each sample and spot were calculated in triplicate. A spot was considered as differential proteins when it came to 5% statistical significance (p < 0.05) in one-way ANOVA.

Gao X., Zhao D., Wang L., Cui Y., Wang S., Lv M., Zang F, & Dai R. (2021). Proteomic Changes in Sarcoplasmic and Myofibrillar Proteins Associated with Color Stability of Ovine Muscle during Post-Mortem Storage. Foods, 10(12), 2989.

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Two-Dimensional Protein Separation

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The procedure was performed using components of ReadyPrep 2-D Starter Kit (cat. 163-2105, Bio-Rad) accordingly to the manufacturer protocol. Firstly, the samples were desalted (final ~10mM) upon dialysis to low-salt buffer and concentration on Amicon^™ centrifugal filters. Next, samples were diluted in rehydration buffer up to 4-fold and loaded on the ReadyStrip^™ IPG Strips 3-10 (cat. 163-2000, Bio-Rad). After overnight rehydratation, the strips were run on the PROTEAN IEF (Bio-Rad) using the protocol suitable for 7cm-long strip. When the run was completed, the gel was washed with the provided equilibration solutions, briefly washed in the cathode buffer, and transferred to 10% SDS-PAGE gel without the stacking part. ReadyPrep^™ Overlay Agarose (163-2111) was poured on the top, and after solidification, the gel was run using standard parameters and imaged with Coomassie blue staining.

Wysocka A., Jagielska E., Łężniak Ł, & Sabała I. (2021). Two New M23 Peptidoglycan Hydrolases With Distinct Net Charge. Frontiers in Microbiology, 12, 719689.

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Protein Extraction and Separation Protocols

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Protein concentrations were estimated using a quick-start Bradford kit [23 (link)]. The required quantities of proteins were precipitated to remove interfering materials using a 2D-clean up kit (Bio-Rad) and resolubilised in rehydration buffer (7 M urea, 2 M thiourea, 2% CHAPS, 0.2% w/v ampholytes, 50 mM DTT and 0.004% bromophenol blue). An IPG-strip (Bio-Rad) of required pI/length was passively rehydrated with the resolubilised sample for 18 hr and focused using PROTEAN-IEF (Bio-Rad). The focused IPG-strips were equilibrated for 30 min each using equilibration buffer-I (6 M urea, 2 M thiourea, 0.375 M Tris–HCl, pH 8.8, 20% glycerol, 2% SDS, 0.005% bromophenol blue and 2% DTT) followed by buffer-II (2.5% iodoacetamide in place of DTT). The strips were then sealed on top of a gradient polyacrylamide gel with 0.5% agarose in electrophoresis buffer and separated using Bio-Rad gel apparatus [21 (link),24 (link)].
Gels were silver stained for visualization and were subjected to MALDI-compatible colloidal coomassie/bio-safe coomassie staining for sequencing [25 (link),26 (link)]. The gels were scanned using a Pro-Pic-II (Genomic solution, USA) image scanner and stored in 1% acetic acid at a temperature of 4°C.

Bag A.K., Saha S., Sundar S., Saha B., Chakrabarti A, & Mandal C. (2014). Comparative proteomics and glycoproteomics of plasma proteins in Indian visceral leishmaniasis. Proteome Science, 12, 48.

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Fluorescent Labeling of GBA Proteins

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ABP-labeled GBA was precipitated by TCA, re-hydrated (30 mM Tris, 7.7 M urea, 2.2 M thiourea, pH 8.9, 4% (w/v) CHAPS, 0.5% (v/v) Destreak agent and 2% (v/v) IPG buffer), soaked overnight into pH 3−10 nonlinear strips (BioRad). Iso-electric focusing with standard program (0.1 min at 50 V; 30 min to 200 V; 30 min at 200 V; 30 min to 400 V; 30 min at 400 V; 30 min to 600 V; 30 min at 600 V; 60 min to 3,500 V; 240 min at 3,500 V; 10 min to 200 V; hold at 200 V, on Protean IEF (BioRad)) and proteins subsequently separated by SDS-PAGE. One-dimensional SDS-PAGE followed by fluorescent scanning and Coomassie Brilliant Blue (CBB) staining was exactly performed as described earlier [50 (link)]. Fluorescence controls were either based on excess imiglucerase labeled with 50 fmol green β-epoxide ABP 5 and red 6, denatured, mixed and loaded on gel as control standard, or based on excess imiglucerase labeled with 100 fmol green β-epoxide ABP 5 and an equimolar amount of velaglucerase labeled with 100 fmol red 6, denatured, mixed and loaded on gel as control standard.

Kallemeijn W.W., Scheij S., Hoogendoorn S., Witte M.D., Herrera Moro Chao D., van Roomen C.P., Ottenhoff R., Overkleeft H.S., Boot R.G, & Aerts J.M. (2017). Investigations on therapeutic glucocerebrosidases through paired detection with fluorescent activity-based probes. PLoS ONE, 12(2), e0170268.

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Isoelectric Focusing of Protein Samples

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A protein sample (250 µg) was mixed with rehydration buffer (RB): 7M urea, 2M thiourea, 4% CHAPS, 0.05% triton X100, 0.5% ampholytes (IPG buffer 4–7 GE) and adjusted to the correct volume to rehydrate 18 cm strip (here, 320 µL). Strips were then placed acrylamide face down in the focusing tray equipped with platinum electrode embedded into the running tray (Protean IEF, Bio-Rad, Hercules, CA, USA) and passively re hydrated at 20°C without electricity for 16 h, and then actively rehydrated at 50 V during 9 h, as previously described [27 (link),35 (link),36 (link)]. During protein focalization, small Electrode wicks were placed between acrylamide and electrode. These paper wicks (Ref 1654071, Electrode wicks, Bio-Rad, Hercules, CA, USA) were, in advance, soaked with water in order to absorb salts and other contaminant species during active rehydration. The IPG strips were then focused according to the following program: 500 V for 1 h, a linear ramp to 1000 V for 1 h, a linear ramp to 10000 V for 33 KV-1 h, and finally 10000 V for 24 KV-1 h.

Tudor M., Gilbert A., Lepleux C., Temelie M., Hem S., Armengaud J., Brotin E., Haghdoost S., Savu D, & Chevalier F. (2021). A Proteomic Study Suggests Stress Granules as New Potential Actors in Radiation-Induced Bystander Effects. International Journal of Molecular Sciences, 22(15), 7957.

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Proteomic Analysis of Mycobacterium smegmatis

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Whole cell extracts were prepared from 100 mL of cultures of each M. smegmatis strain in the log-phase. Bacterial pellets were washed twice and centrifuged (15°C, 8000 rpm, 15 min) in 0.02 M PBS (pH 7.4). The cells were sonicated in 500 μL of 0.02 PBS (0.02 M supplemented with 1 mM PMSF and 10 mM EDTA) and centrifuged at 12,000 rpm for 20 min at 4°C. The cells were treated with lysis buffer (8 M urea, 2 M thiourea, 140 mM DTT, 0.5% Biolyte pH 4-7, and 4% CHAPs) for 1 h and centrifuged (4°C, 12,000 rpm, 15 min). The supernatant was harvested and the protein concentration was estimated using the Bradford assay. Total protein (80 μg) were loaded on linear pH 4-7 IPG strips (Bio-Rad, United States) that were allowed to rehydrate at 50 V for 13 h. Isoelectric focusing was carried out using a Protean IEF (Bio-Rad, United States). The proteins were separated using 2-dimensional SDS-PAGE (12% gels) and stained with silver nitrate, as reported previously (Scheler et al., 1998 (link); Gharahdaghi et al., 1999 (link)). The gels were scanned using Molecular Image FX (Bio-Rad, United States) and analyzed with the PD Quest 6.0 software (Bio-Rad, United States). The proteins of interest were excised from the silver-stained 2D gels and identified by MALDI-TOF-MS (Fu and Fu-Liu, 2007 (link)).

Liu D., Hao K., Wang W., Peng C., Dai Y., Jin R., Xu W., He L., Wang H., Wang H., Zhang L, & Wang Q. (2017). Rv2629 Overexpression Delays Mycobacterium smegmatis and Mycobacteria tuberculosis Entry into Log-Phase and Increases Pathogenicity of Mycobacterium smegmatis in Mice. Frontiers in Microbiology, 8, 2231.

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