Two-dimensional electrophoresis of proteins was performed in accordance with the method of O'Farrell (1975 (link)). An immobiline dry strip gel (17 cm, pH 4-7; Bio-Rad, USA) was rehydrated at 20°C for 14 h in 200 μl of sample containing 500 μg of protein. Isoelectric focussing (first-dimensional separation) was carried out in a PROTEAN IEF apparatus (BioRad, USA). The voltages applied were 250 V for 1 h, 500 V for 1 h, 1000 V for 2 h, 2000 V for 2 h, linear increase of 8000 V for 18 h and 500 V for 1 h. After the completion of IEF, strips were subjected to reduction for 15 min by the reduction buffer and then to alkylation buffer for 15 min as mentioned in Bagheri et al. (2015 (link)). The SDS-PAGE was carried out in a Dodeca cell (PROTEAN plus, Bio-Rad, USA) for the second-dimensional separation of focussed proteins using 12% SDS at a constant voltage of 100 V. The gels were stained by colloidal Coommassie brilliant blue dye followed by destaining.
Protean ief apparatus
Manufactured by Bio-Rad
Sourced in United States
The PROTEAN IEF apparatus is a laboratory equipment designed for isoelectric focusing (IEF) separation of proteins. It provides a controlled environment for the separation of proteins based on their isoelectric point. The apparatus includes components necessary for the IEF process, such as a focusing tray, electrodes, and a power supply.
Automatically generated - may contain errors
Lab products found in correlation
5 protocols using protean ief apparatus
1
Two-Dimensional Protein Electrophoresis Protocol
2
Isoelectric Focusing of Protein Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The nonlinear 7 cm long immobilised pH gradient (IPG) strips with pH 3–10 gradients were hydrated with 125 μL of the above-treated protein sample (100 μg). The IEF was then carried out by using Protean IEF apparatus (Bio-Rad, USA) with the following program: desalting at 250 V for 15 min, ramping up the voltage to 4000 V by a linear gradient for 2 h, keeping 4000 V constant for a total of 37500 Vh and then terminating the isoelectric focusing or holding at 500 V until the termination. The temperature of the IEF apparatus during isoelectric focusing was 17°C. Upon completion of the isoelectric focusing, the IPG strips were immediately subjected to the second dimension.
3
Protein Extraction and 2D-PAGE Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The extraction of protein in the latest fully expanded leave for 2 DE was performed following the trichloroacetic acid (TCA) acetone precipitation method described by Ding et al. [22 (link)].
Immobiline DryStrip gels (117 cm length: Bio-Rad) were used for first dimension isoelectrofocusing (IEF) at pH 4 to 7. Rehydration and focus were performed using PROTEAN IEF apparatus (Bio-Rad) at 50 μA per strip at 20 °C, using the following programme: 12 h of rehydration at 50 V in rehydration buffer (7 M urea, 2 M thiourea, 4 % (w/v) CHAPS, 0.5 % (v/v) IPG buffer, 10 mM DTT, and 0.1 % bromophenol blue), 1 h at 500 V, 1 h at 1 000 V, 2 h at 8 000 V, and 85 000 V · hours at 8 000 V. After dimension isoelectrofocusing, strips were equilibrated for 15 min in SDS equilibration buffer solution (6 M urea, 37.5 mM Tris-HCl (pH 6.8), 20 % (v/v) glycerol, 2 % (w/v) SDS, and 1 % (w/v) DTT), followed by equilibration with a buffer containing 135 mM iodoacetamide for 15 min. After equilibration, proteins were distributed in the second dimension (SDS-PAGE) using 10 % polyacrylamide gels (250 × 200 × 1 mm), and the gels were stained with silver nitrate solution.
Immobiline DryStrip gels (117 cm length: Bio-Rad) were used for first dimension isoelectrofocusing (IEF) at pH 4 to 7. Rehydration and focus were performed using PROTEAN IEF apparatus (Bio-Rad) at 50 μA per strip at 20 °C, using the following programme: 12 h of rehydration at 50 V in rehydration buffer (7 M urea, 2 M thiourea, 4 % (w/v) CHAPS, 0.5 % (v/v) IPG buffer, 10 mM DTT, and 0.1 % bromophenol blue), 1 h at 500 V, 1 h at 1 000 V, 2 h at 8 000 V, and 85 000 V · hours at 8 000 V. After dimension isoelectrofocusing, strips were equilibrated for 15 min in SDS equilibration buffer solution (6 M urea, 37.5 mM Tris-HCl (pH 6.8), 20 % (v/v) glycerol, 2 % (w/v) SDS, and 1 % (w/v) DTT), followed by equilibration with a buffer containing 135 mM iodoacetamide for 15 min. After equilibration, proteins were distributed in the second dimension (SDS-PAGE) using 10 % polyacrylamide gels (250 × 200 × 1 mm), and the gels were stained with silver nitrate solution.
4
Comprehensive Proteome Analysis of Rice Genotypes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Based on the physiological response information of the two rice genotypes exposed to the nitrogen treatment at the pot, 2DE of proteins was performed to separate the leaf proteins at 10 DAF using the isoelectric focusing (IEF) strip gels (17 cm, pH 4–7; Bio-Rad, Hercules, CA, USA) for the first dimension. For IEF, an immobiline dry strip gel was rehydrated at 20 °C for 14 h and 1.3 mg protein was loaded in each strip. Protean IEF apparatus (Bio-Rad, USA) was used to do IEF. The voltages applied were at a gradient of 500 V for 1 h; gradient of 1000 V for 2 h; gradient of 8000 V for 3 h; held at 8000 V for 3 h; and then at a gradient of 1000 V for 24 h, as mentioned in [45 (link)]. After completing the IEF, the strips were further exposed to an equilibration buffer. Equilibration buffer I (65 mM DTT) was used for 15 min shaking. Iodoacetamide (2.5% (w/v) was used and kept shaking for 15 min. The second-dimensional separation was carried out on SDS-PAGE containing 12% (v/v) polyacrylamide gels in 2D-Electrophoresis SDS-PAGE Apparatus (GE) at 15 mA current per gel until completion of electrophoresis. The gels were stained with Colloidal Coomassie Blue G-250 for 12 h followed by destaining. GE Image scanner III was used to scan the obtained protein gels and Imagemaster 5.0 software was used to identify comparative protein spots.
5
Leaf Proteome Profiling by 2D-Electrophoresis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Two-dimensional electrophoresis of proteins was performed to resolve the leaf proteome. In the first dimensional run, IPG strips (24 cm, pH 3–10, NL; Bio-Rad, USA) were used. From each treatment 500 μg protein (in 400 μl rehydration buffer) was loaded through passive rehydration at 20°C for 14 h. Isoelectric focussing was carried out in a PROTEAN IEF apparatus (Bio-Rad, USA). The voltages applied were 250 V for 1 h, 500 V for 1 h, 1000 V for 2 h, 2000 V for 2 h, linear increase of 8000 V and running till achieving 80,000 Vh, followed by a slow ramping of 500 V for 1 h. After the completion of IEF, the strips were exposed to reduction buffer for 15 min and then to alkylation buffer for 15 min. The SDS-PAGE was carried out in a Dodeca cell (Bio-Rad PROTEAN plus, USA) for separation of focussed proteins, using 12% SDS at a constant voltage of 250 V. The gels were stained with colloidal Coommassie brilliant blue dye and then destained with ultrapure water.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!