Ipgbox

Manufactured by GE Healthcare

Sourced in United Kingdom

The IPGbox is a laboratory equipment product from GE Healthcare. It is designed to provide a controlled environment for protein purification and analysis. The IPGbox functions as an isoelectric focusing unit, a key step in two-dimensional gel electrophoresis. The device creates a stable pH gradient that enables the separation and identification of complex protein mixtures.

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Lab products found in correlation

Immobiline drystrip, by GE Healthcare (3 mentions) Ettan ipgphor 3 focusing unit, by GE Healthcare (2 mentions) Ettan ipgphor 3 ief system, by GE Healthcare (2 mentions) Bio lyte 3 10 ampholyte, by Bio-Rad (2 mentions) Ettan ipgphor, by GE Healthcare (2 mentions) Ettan daltsix large vertical system, by GE Healthcare (2 mentions) Immobiline dry strips ph 3 10 nl, by GE Healthcare (1 mentions) Destreak, by GE Healthcare (1 mentions) 2 d clean up kit, by GE Healthcare (1 mentions) Ettan ipgphor 3 isoelectric focusing unit, by GE Healthcare (1 mentions) Ettan ipgphor 3, by GE Healthcare (1 mentions) High resolution scanner, by Epson (1 mentions) Pdquest advanced 2 d analysis software, by Bio-Rad (1 mentions) Destreak rehydration solution, by GE Healthcare (1 mentions) Agarose sealing buffer, by Bio-Rad (1 mentions) Ipg buffer ph 4 7, by GE Healthcare (1 mentions) Iodoacetamide, by Merck Group (1 mentions) Urea, by Merck Group (1 mentions)

11 protocols using ipgbox

Two-Dimensional Gel Electrophoresis of Spermatozoan Proteins

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The gels were made in triplicate per animal, totalling 15 profile maps. Spermatozoan proteins (250 µg) were solubilized in rehydration buffer (7 M urea, 2 M thiourea, 65 mM DTT, 1% (w/v) CHAPS, 0.5% (v/v) ampholytes, and trace amounts of bromophenol blue. The samples were applied to an IPGBox (GE Healthcare) and were incubated on 13 cm IPG strips with a linear pH gradient (pH 4-7) for 16 hours.
Isoelectric focusing was performed using an Ettan™ IPGPhor 3™ Focusing Unit (GE Healthcare) under the following conditions: step 1, 500 V for 30 minutes; step 2, 4,000 V for 2.5 hours; and step 3, 8,000 V until 18,000 total volt-hours is reached. The strips were then stored at -80 °C for later use. The strips were equilibrated in an equilibrium solution (50 mM Tris, 30% glycerol, 6 M urea, 2% SDS and trace amounts of bromophenol blue) with 1% (w/v) DTT for 15 minutes. Strips were then immediately incubated in an equilibrium solution containing 3% (w/v) iodoacetamide for 15 minutes. Finally, the proteins were separated along the second dimension using 12.5% polyacrylamide gels in the presence of SDS with 15 mA/gel for 15 minutes and 50 mA/gel for 4-8 hours.

Pinto T.M., Moreira R.F., Matos M.N., Soares V.V., Aguiar M.V., de Aragão P.D., Alves JG F.i.l.h.o., Moreno F.B., Monteiro-Moreira A.C., Costa C.R., de Lima JL F.i.l.h.o., Eloy A.M, & da Cunha R.M. (2019). Evaluation of the proteomic profiles of ejaculated spermatozoa from Saanen bucks ( Capra hircus ). Animal Reproduction, 16(4), 902-913.

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2D Electrophoresis of OMV Bp Proteins

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2DE was performed using the Ettan IPGphor 3 isoelecteric focusing system (GE Healthcare, USA). The incubation of the samples for 2DE was performed after sample preparation and IR-labeling. About 350 μg OMV Bp proteins was incubated overnight at laboratory temperature in DeStreak Rehydration solution (final volume = 250 μL) which contained 7 M urea, 2 M thiourea, 100 mM, 4% CHAPS, 0.2% carrier ampholyte, 0.0002% bromophenol blue, 4 mg of 50 mM dithiothreitol (DTT) and 1.5 μL of IPG buffer (pH 3-10 NL) (GE Healthcare). Immobiline Dry Strips pH 3-10 NL, 7 cm (GE Healthcare) were rehydrated with protein sample in an IPGbox (GE Health-care). Isoelectric focusing (IEF) was performed in an Ettan IPGphor 3 IEF system (GE Healthcare) according to the following conditions: 300 V for 30 min, 1000 V for 30 min, 5000 V for 90 min, finally 5000 V for 8 KVh, at 20 °C and a current limit of 50 μA per strip. After IEF, strips were equilibrated in 3 mL of equilibration buffer (150 mM Tris-HCl, pH 8.8, 6 M urea, 50% glycerol, 20% SDS, and bromophenol blue) with DTT (2%) for 15 min at RT, followed by 3 mL of equilibration buffer with iodoacetamide (2.5%) for 15 min at RT. The proteins were separated in second dimension on 10% SDS -PAGE in a vertical electrophoretic dual gel system and were visualized by Coomassie (R-250) staining method (13 ).

Badamchi A., Bahrami F., Tasbiti A.H., Yari S., Shafiei M., Shahcheraghi F, & Siadat S.D. (2020). Immuno-proteomics analysis between OMV of vaccine and dominant wild type strains of Bordetella pertussis in Iran. Iranian Journal of Microbiology, 12(2), 77-88.

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1D Electrophoresis of Protein Samples

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For one dimensional (1D) electrophoresis, a 125 μL solubilizing buffer containing 70 μg protein was rehydrated passively for 15 h in 7 cm immobilized pH gradient (IPG) strip (pH 4–7) in the IPGbox (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The focusing was carried out in the Ettan IPGphor 2 isoelectric focusing (IEF) unit (GE Healthcare, Little Chalfont, Buckinghamshire, UK) at 20°C with 50 μA per strip on the following conditions; 300 V for 0:30 (h:min) (Step and Hold), 1000 V for 0:30 (h:min) (Gradient), 5000 V for 1:30 (h:min) (Gradient), and 5000 V for 0.36 (h:min) (Step and Hold). Total time taken until final volt reaches 8.0 KVh was 3.06 (h:min).

Soundararajan P., Manivannan A., Cho Y.S, & Jeong B.R. (2017). Exogenous Supplementation of Silicon Improved the Recovery of Hyperhydric Shoots in Dianthus caryophyllus L. by Stabilizing the Physiology and Protein Expression. Frontiers in Plant Science, 8, 738.

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2D Gel Electrophoresis Proteomics Workflow

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2D gel electrophoresis experiments were performed based on previous reports39 with some modifications. Fifty micrograms of proteins from fly heads were precipitated using the 2D clean-up kit (GE Healthcare). The resulting pellet was resuspended in 2D buffer (7 M urea, 2.5 M thiourea, 4% CHAPS) by sonication and supplemented with 0.5% IPG buffer 3–11 (GE Healthcare), 1% destreak (GE Healthcare) and traces of Bromophenol blue. Following overnight incubation with the protein samples in an IPG box (GE Healthcare), rehydrated Immobiline Drystrips (pH 3–11 NL, 11 cm) were placed into the Ettan IPGphor 3 Isoelectric Focusing Unit (GE Healthcare) and covered with mineral oil. The isoelectrofocalisation was performed following the manufacturer’s instructions, after which the strips were incubated in equilibration buffer (6 M urea, 30% glycerol, 2% SDS, 50 mM Tris-HCl pH 6.8, traces of bromophenol blue) supplemented first with DTT (10 mg/mL) for 15 min and with iodoacetamide (50 mg/mL) for another 15 min. Strips were then placed on top of 10% Bis-Tris IPG+1 Criterion gels (Biorad) for SDS-PAGE and proteins were subsequently transferred to 0.45 μm nitrocellulose membranes for antibody staining.

Burnouf S., Grönke S., Augustin H., Dols J., Gorsky M.K., Werner J., Kerr F., Alic N., Martinez P, & Partridge L. (2016). Deletion of endogenous Tau proteins is not detrimental in Drosophila. Scientific Reports, 6, 23102.

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Sperm Proteome Profiling by 2D-PAGE

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Para as análises proteômicas foram feitos dois géis 2D para cada tratamento.
Spermatozoid proteins (250 µg) were solubilized in rehydration buffer (7 M urea,
2 M thiourea, 65 mM DTT, 1% (w/v) CHAPS, 0.5% (v/v) ampholytes, and trace amounts of bromophenol
blue). The samples were applied to an IPGBox (GE Healthcare) and incubated on 7-cm immobilized
pH gradient (IPG) strips with a linear pH gradient (pH 4-7) for 16 h.
Isoelectric focusing was performed using an Ettan™ IPGPhor 3™ Focusing Unit
(GE Healthcare) under the following conditions: step 1, 500 V for 30 min; step 2, 4000 V for 2.5
hours; and step 3, 8000 V until reaching 18,000 total volt-hours. The strips were then stored
at -80°C for later use. The strips were equilibrated in an equilibrium solution (50
mM Tris, 30% glycerol, 6 M urea, 2% SDS and trace amounts of bromophenol blue) with 1% (w/v) DTT
for 15 min. The samples were then immediately incubated in an equilibrium solution containing
3% (w/v) iodoacetamide for 15 min. Finally, the proteins were separated along the second dimension
using 12.5% polyacrylamide gels in the presence of SDS with 15 mA/gel for 15 min and 50 mA/gel
for 4-8 hours.

Moreira R.F., Matos M.N., Alves JG F.i.l.h.o., do Valle R.V., Eloy A.M., Pinto T.M., Machado SP J.u.n.i.o.r., Costa C.R., de Lima JL F.i.l.h.o., Lima J.P, & da Cunha R.M. (2018). Diversity of ejaculated sperm proteins in Moxotó bucks (Capra hircus ) evaluated by multiple extraction methods. Animal Reproduction, 15(1), 84-92.

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Protein Separation and Quantification

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For the first dimension, isoelectric focusing (IEF) of proteins was performed based on the method described by Gorg et al. [65 (link)] with some modifications. The immobilized pH gradient (IPG) strips (18 cm, pH 3–10 NL) were passively rehydrated with 500 μg of protein in a 360 μL rehydration buffer in the IPGbox (GE Healthcare, Little Chalfont, Buckinghamshire, UK) for 12 h. The focusing was performed in the Ettan IPGphor 3 (GE Healthcare, UK) at 20 °C with 50 mA per strip under the following conditions: 30 V for 2 h, 200 V for 1 h, 500 V for 1 h, 3000 V for 1 h, gradient from 3000 V to 8000 V within 30 min, and 8000 V for 3 h. After the first dimensional IEF, the IPG strips were equilibrated according to the method of Gorg et al. [65 (link)]. The second dimensional electrophoresis was carried out in 12% SDS-PAGE, then the gels were stained with Coomassie brilliant blue (CBB) according to R-250/G-250 = 4:1. The images were taken with a high-resolution scanner (Epson, Long Beach, USA). The abundant difference (2-fold, p < 0.05) of the proteins in the samples were calculated with the PDQuest Advanced 2-D Analysis software (version 8.01, Bio-Rad Laboratories, Hercules, CA, USA) based on Student’s t-test.

Li Y., Hu J., Wei H, & Jeong B.R. (2020). A Long-Day Photoperiod and 6-Benzyladenine Promote Runner Formation through Upregulation of Soluble Sugar Content in Strawberry. International Journal of Molecular Sciences, 21(14), 4917.

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Two-Dimensional Gel Electrophoresis of B. pertussis Lysate

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After the sample preparation, 50 μg B. pertussis B1917 lysate was incubated for 30 min at RT after adding 10 μl 250 mM DTT, 0.62 μl IPG buffer pH 3–10 non-linear (NL), or IPG buffer pH 4-7 (GE Healthcare, Netherlands), and DeStreak Rehydration Solution (GE Healthcare, Netherlands) to a final volume of 115 μl. Immobiline DryStrips pH 3–10 NL, 7 cm, or pH 4–7, 7 cm (GE Healthcare, Netherlands) were rehydrated with sample in an IPGbox (GE Healthcare, Netherlands). Isoelectric focusing (IEF) was performed in the Ettan IPGphor three IEF system (GE Healthcare, Netherlands) with the following program: 0.5 h gradient to 300 V, 1 h gradient to 1,000 V, 1 h 2,000 V, 1 h 3,000 V, 1 h 4,000 V, and 1 h 5,000 V. After IEF strips were equilibrated 15 min in 3 mL equilibration buffer containing 75 mM Tris-HCl pH 8.8, 6 M Urea (Sigma Aldrich, Merck, Germany), 30% Glycerol, 2% SDS, bromophenol blue and 65 mM DTT, followed by a 15 min equilibration with equilibration buffer containing 54 mM iodoacetamide (Sigma Aldrich, Germany) instead of 65 mM DTT. Strip was placed on a 4–12% NuPAGE bis-tris Zoom gel (Thermo Fisher, Invitrogen, Netherlands) and sealed with Agarose sealing buffer (BioRad, Netherlands). Proteins were separated in MES running buffer in a Xcell surelock minicell electrophoresis system for 50 min at 200 V. Gels were stained with Coomassie or used for Western blot.

Raeven R.H., van der Maas L., Pennings J.L., Fuursted K., Jørgensen C.S., van Riet E., Metz B., Kersten G.F, & Dalby T. (2019). Antibody Specificity Following a Recent Bordetella pertussis Infection in Adolescence Is Correlated With the Pertussis Vaccine Received in Childhood. Frontiers in Immunology, 10, 1364.

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Two-Dimensional Protein Separation Protocol

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For each strip, 150 μg of protein (50 μg from each dye) were loaded along with rehydration buffer (8 M urea, 2% CHAPS, 50 mM DTT, 0.001% bromophenol blue, 0.5% Bio-lyte 3/10 ampholyte (Bio-Rad Laboratories, Hercules, California, USA) to complete 450 μl. Passive rehydration was conducted for 15 h on 24 cm Immobiline™ Drystrips (GE Healthcare, Little Chalfont, UK) with linear pH 4–7, on an IPG Box (GE Healthcare, Little Chalfont, UK). Following, isoelectric focusing (IEF) was performed in 5 steps: 500 V gradient 1 h, 500 V step-n-hold 1 h, 1000 V gradient 1 h, 8000 V gradient 3 h and 8000 V step-n-hold 5 h40 for a total of 60.000 Vhr using Ettan IPGphor at 20 °C (GE Healthcare, Little Chalfont, UK). Focused strips were reduced and alkylated with 6 ml of equilibration buffer (50 mM Tris-HCl pH 8.8, 6 M urea, 30% (v/v) glycerol and 2% SDS) with 1% (w/v) dithiothreitol (DTT) or 2.5% (w/v) iodoacetamide (IAA) respectively for 15 min each, in constant agitation. Strips were then loaded onto 12.5% Tris-HCl SDS-PAGE gels and ran in an Ettan DALTsix Large Vertical System (GE Healthcare, Little Chalfont, UK) at 10 mA/gel for 1 h followed by 60 mA/gel until the bromophenol blue line reaches the end of the gel, using a standard Tris-Glycine-SDS running buffer.

Raposo de Magalhães C., Schrama D., Farinha A.P., Revets D., Kuehn A., Planchon S., Rodrigues P.M, & Cerqueira M. (2020). Protein changes as robust signatures of fish chronic stress: a proteomics approach to fish welfare research. BMC Genomics, 21, 309.

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2D Gel Electrophoresis Protein Separation

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Labeled proteins from each treatment plus 50 µg of internal standard were mixed together and rehydration buffer (6M urea, 2M thiourea, 4% CHAPS, 0.02 % (w/v) DTT, 0.002% bromophenol blue, 0.5% (v/v) IPG buffer pH 4-7) was added to complete 450 µl. Rehydration was performed passively for 14 hours using IPG box (GE Healthcare) on 24 cm Immobiline™ DryStrips (GE Healthcare) with linear pH 4-7, continued by isoelectric focusing (IEF) in 5 steps: at 500V gradient 1 hr, at 500V step-n-hold 1 hr, at 1000V gradient 1 hr, at 8000V gradient 3 hrs, and at 8000V stepn-hold for a total of 60000 Vhr. Before second dimension, strips were reduced and alkylated using 6 ml of an equilibration buffer (1.5M Tris-HCl pH 8.8, 6M urea, 30% (v/v) glycerol, 0.007M SDS, a few grains of bromophenol blue) with 1% (w/v) DTT or 2.5% (w/v) iodoacetamide respectively for 15 min each. Strips with the samples and internal standard were loaded onto 12.5% Tris-HCl SDS-PAGE gels and run in an Ettan DALTsix Vertical System at 10 mAgel -1 for 1 hour followed by 60 R e v i e w C o p y mAgel -1 using a standard Tris-Glycine-SDS running buffer, until the bromophenol blue line reaches the end of the gel.

Moreira M., Schrama D., Soares F., Wulff T., Pousão-Ferreira P, & Rodrigues P. (2017). Physiological responses of reared sea bream (Sparus aurata Linnaeus, 1758) to an Amyloodinium ocellatum outbreak. Journal of fish diseases, 40(11).

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2D-DIGE Protein Separation Protocol

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Protein separation was achieved by the two-dimensional difference gel electrophoresis (2D-DIGE) method, as follows. DIGE minimal labeling was performed as described in (Raposo de Magalhães et al., 2020b) . Passive rehydration was carried out for 18 h on 24 cm Immobiline TM Drystrips (GE Healthcare) with linear pH 4-7, on an IPG Box (GE Healthcare). A total of 150 µg of protein (50 µg from each dye) were loaded onto each strip, along with a rehydration buffer [8 M urea, 2% CHAPS detergent, 50 mM dithiothreitol (DTT), 0.001% bromophenol blue, 0.5% Bio-lyte 3/10 ampholyte (Bio-Rad)] to fulfill 450 µl. Isoelectric focusing (IEF) was performed in 4 steps: 250 V gradient 4 h, 1000 V gradient 6 h, 8000 V gradient 3 h 40 min, 8000 V step-n-hold 3 h 20 min for a total of 50,000 Vhr using the Ettan IPGphor at 20 • C (GE Healthcare). Focused strips were reduced and alkylated with 6 ml of equilibration buffer (50 mM Tris-HCl pH 8.8, 6 M urea, 30% (v/v) glycerol and 2% SDS) with 1% (w/v) DTT or 2.5% (w/v) iodoacetamide (IAA) respectively for 15 min each, in constant agitation. Strips were then loaded onto 12.5% Tris-HCl SDS-PAGE in-house gels and ran in an Ettan DALTsix Large Vertical System (GE Healthcare), using a standard Tris-Glycine-SDS running buffer, at 10 mA/gel for 1 h followed by 60 mA/gel until the bromophenol blue line reaches the end of the gel.

Magalhães C.R.d., Schrama D., Nakharuthai C., Boonanuntanasarn S., Revets D., Planchon S., Kuehn A., Cerqueira M., Carrilho R., Farinha A.P., & Picard B.B. (2021). Metabolic Plasticity of Gilthead Seabream Under Different Stressors: Analysis of the Stress Responsive Hepatic Proteome and Gene Expression. Frontiers in Marine Science, 8.

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