Two dimensional gel electrophoresis

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Information about Two dimensional gel electrophoresis

Published on May 14, 2016

Author: ShyamKUthaman

Source: slideshare.net

1. TWO-DIMENSIONAL GEL ELECTROPHORESIS (2DGE) Shyam K U MFSc (AAHM) student ICAR-CIFE, Mumbai, India INTRODUCTION Electrophoresis is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. It is an analytical method frequently used in molecular biology and medicine. It is applied for the separation and characterization of proteins, nucleic acids and subcellular-sized particles like viruses and small organelles. Its principle is that the charged particles of a sample migrate in an applied electrical field. If conducted in solution, samples are separated according to their surface net charge density. Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA, and proteins) and their fragments, based on their size and charge. There are two types of gel electrophoresis – one dimensional and two dimensional 2-D electrophoresis is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells, tissues, or other biological samples Two-dimensional electrophoresis was first introduced by O’Farrell and Klose in 1975.This 2DGE arises from a question that how we can separate two proteins of having the same molecular mass because this two protein together will give one band after the SDS-PAGE. So we cannot distinguish these two proteins. This technique separates proteins according to two independent properties in two discrete steps The first-dimension step, isoelectric focusing (IEF), separates proteins according to their isoelectric points (pI); the second-dimension step, sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), separates proteins according to their molecular weights. Each spot on the resulting two- dimensional .gel potentially corresponds to a single protein species in the

2. sample. Thousands of different proteins can thus be separated, and information such as the protein pI, the apparent molecular weight, and the amount of each protein can be obtained. In the original technique, the first dimension separation was performed in carrier-ampholyte-containing polyacrylamide gels cast in narrow tubes. carrier-ampholyte-generated pH gradients have been replaced with immobilized pH gradients, and tube gel replaced with gel supported by a plastic backing. Immobiline DryStrip gels offer a marked improvement over tube gels using carrier ampholyte–generated pH gradients. When Immobiline DryStrip gels are used for the first-dimension separation, the resultant 2- D spot maps provide superior results in terms of resolution and reproducibility PRINCIPLE FIRST STEP:- FIRST DIMENSIONAL ISOELECTRIC FOCUSING (IEF) Isoelectric Focusing is an electrophoretic method that separates proteins according to their isoelectric points (pI). Proteins are amphoteric molecules; they carry either positive, negative, or zero net charge, depending on the pH of their surroundings The net charge of a protein is the sum of all the negative and positive charges of its amino acid side chains and amino- and carboxyl-termini. The isoelectric point (pI) is the specific pH at which the net charge of the protein is zero. Proteins are positively charged at pH values below their pI and negatively charged at pH values above their isoelectric point. The presence of a pH gradient is critical to the IEF technique. In a pH gradient and under the influence of an electric field, a protein will move to the position in the gradient where its net charge is zero. A protein with a net positive charge will migrate toward the cathode, becoming progressively less positively charged as it moves through the pH gradient until it reaches its pI. A protein with a net negative charge will migrate toward the anode, becoming less negatively charged until it also reaches zero net charges. If a protein should diffuse away from its pI, it

3. immediately gains charge and migrates back. This is the focusing effect of IEF, which concentrates proteins at their pI’s and allows proteins to be separated on the basis of very small charge differences The resolution is determined by the slope of the pH gradient and the electric field strength. IEF is therefore performed at high voltages (typically in excess of 1000 V). When the proteins have reached their final positions in the pH gradient, there is very little ionic movement in the system, resulting in a very low final current (typically in the microamp range). IEF of a given sample in a given electrophoresis system is generally performed for a constant number of Volt-hours (Volt-hour [Vh] being the integral of the volts applied over the separation time). IEF performed under denaturing conditions gives the highest resolution and the sharpest results. Complete denaturation and solubilization are achieved with a mixture of urea, detergent, and reductant, ensuring that each protein is present in only one conformation with no aggregation, therefore minimizing intermolecular interactions.

4. 2. SECOND STEP:- 2-DIMENSION SDS-PAGE SDS-PAGE is an electrophoretic method for separating polypeptides according to their molecular weights (Mr). The technique is performed in polyacrylamide gels containing sodium dodecyl sulfate (SDS). The intrinsic electrical charge of the sample proteins is not a factor in the separation due to the presence of SDS in the sample and the gel. The polyacrylamide gel is cast as the separating gel (resolving or running gel), topped by usually the stacking gel, and secured in an electrophoresis apparatus. But here the IPG strip is placing instead of stacking gel. The proteins are get denatured and solubilized in the presence of 0.1% SDS upon heating. When the SDS in solution in water, forms globular micelles composed of 70–80 molecules with the dodecyl hydrocarbon moiety in the core and the sulfate head groups in the hydrophilic shell. SDS and proteins form complexes with a necklace-like structure composed of protein-decorated micelles connected by short flexible polypeptide segments (77). The result of the necklace structure is that large amounts of SDS are incorporated in the SDS-protein complex in a ratio of approximately 1.4 g SDS/g protein. SDS masks the charge of the proteins themselves and the formed anionic complexes have a roughly constant net negative charge per unit mass In addition to this, by adding thiols such as Dithiothreitol (DTT) or 2- mercaptoethanol (2-ME), reduces disulfide bonds, ensuring that only single polypeptides are resolved. This will lead to a constant charge to mass ratio and identical charge densities for the denatured proteins. The constant charge to mass ratio means that the protein migrates through the electrophoresis gel based on size – the larger the protein, the more it interacts with the gel pores and the more slowly it migrates. Thus, the SDS- Protein complex are migrated in the gel according to size, not charge.

5. Polyacrylamide gels form through the copolymerization of monomeric acrylamide to form the long chains and crosslinking of those chains by bisacrylamide monomer (N,N’–methylene bisacrylamide). The polymerization agent used is Ammonium Per Sulfate (APS) 10% and N,N,N’,N’–tetramethylene diamine(TEMED) as a catalyst for the formation of free radicles from APS. This should be added just before casting the gel. Most proteins are resolved on polyacrylamide gels containing from 5% to 15% acrylamide and 0.2% to 0.5% bisacrylamide (1:37.5). The ratio will increase when the sample contains large proteins and vice-versa PROTOCOL 1. Sample Preparation For eg : Prepare cell lysates with Trizol extraction. Determining Protein Concentration of cell lysates and dilute with Buffer. For aqueous samples, dilute a portion of the protein sample to be analyzed 1:1(v/v) with IEF buffer 2. First dimension Separation: Isoelectric Focusing (IEF) : using Immobilized pH Gradient (IPG) strips Immobilized pH gradients are formed using two solutions, one containing a relatively acidic mixture of acrylamide buffers and the other containing a relatively basic buffer mixture. Added to polyacrylamide gel and cast onto a plastic backing and cut into 3mm wide strips, then dry it. (i) Select an appropriate IPG strip size according to your sample volume (ii) Prepare the strip holder(s) corresponding to the IPG strip length chosen for the experiment. Handle the ceramic strip holder with care, as they are fragile. (iii) Wash strip holder: Rinse each strip holder with ddH2O followed by adding a few drops of Holder Cleaning Solution.

6. Use a toothbrush and vigorous agitation to clean the strip holder. Rinse well with ddH2O. Thoroughly dry the strip holders with lint-free tissue prior to use. (iv) Prepare fresh rehydration solution For example: thaw 945uL rehydration stock (stock in the – 80°C freezer), add 50uL of 1M DTT and 5uL of IPG buffer solution mix well. (v) Prepare the sample and rehydration solution mixture: add appropriate volume of rehydration solution into the sample to make up the total volume indicated in the following table Table 2. Sample and rehydration solution mixture volume per IPG Strip IPG Strip length (cm) Total volume per strip (uL) 7 125 11 200 13 250 18 340 24 450 (vi) Distribute the rehydration-sample solution mixture evenly in the strip holders. Be sure to record the boat number with the sample name. (vii) Remove the protective cover from the IPG strip starting at the positive (acidic, pointed or arrow) end. Position the IPG strip in the re-swelling holder with the gel side down and the positive end of the strip against the sloped end of the slot. (viii) Overlay each IPG strip with Dry Strip Cover Fluid to minimize evaporation and urea crystallization. (ix) Run IPG strip on the IEF apparatus. (x) Once the program is complete, turn off the power and remove your samples.

7. (xi) IPG strips can either be stored in a plastic Petri dish at -80°C (gel side facing up) or directly follow SDS-PAGE 3. Second dimension Separation: SDS-Page (i) Make an SDS-polyacrylamide gel without the upper stack, leaving about 50mm of the room from the top. (ii) If your strips are stored at -80°C, remove the IPG strips from the freezer and allow them to thaw for 10 to 20 minutes prior to equilibration of strips (iii) If your strips are stored at -80°C, remove the IPG strips from the freezer and allow them to thaw for 10 to 20 minutes prior to equilibration of strips (iv) Wash IPG strips with SDS-PAGE running buffer (1x) before loading to the second-dimension gel in order to get rid of the free DTT and IAA. (v) Apply the equilibrated IPG strips to the second-dimension gel between the two glass plates. Make sure the IPG plastic backing is against ONE of the glass plates. (vi) Seal the IPG strip in place: Add about 1mL of agarose sealing mixture over the gel strip and filter paper to completely cover them. Allow 10 to 15 minutes to solidify. (vii) Run the gel: Generally, the higher the voltage, the faster your samples will run through the gel. However, at too high of a voltage melting as well as a poor visual resolution of your gel will occur. (viii) Stain the gel with Coomassie, Silver or SYPRO Ruby staining to determine protein levels or with ProQ Diamond staining to detect phosphorylated proteins 4. Visualization REAGENTS REQUIRED Solution Component Amount (10 mL) Bromophenol blue 100 mg 1% Bromophenol Blue Stock solution:

8. Tris-base 60 mg Deionized water (Milli-Q) Up to 10 mL Rehydration Buffer: Solution Component Amount (10 mL) Urea 4.2 g Thiourea 1.52 g CHAPS 0.4 g 1 M DTT* 500 µL 100% IPG buffer (Ampholines)* 50 µL 1% Bromophenol blue 200 µL Deionized water (Milli-Q) Up to 10 mL *If freezing Rehydration Buffer do not add DTT or IPG buffer until ready to use SDS Equilibration Buffer: Solution Component Amount (200 mL) 1 M Tris, pH 8.8 10 mL Urea 72.07 g 100% Glycerol 60 mL 10% SDS 40 mL 1% Bromophenol Blue 400 µL Deionized water (Milli-Q) Up to 200 mL This is a stock solution. Prior to use DTT or iodoacetamide are added 12% SDS-Polyacrylamide Gel: Solution Component Amount (10 mL) Deionized water (Milli-Q) 3.3 mL 30% Acrylamide 4.0 mL 1.5 M Tris, pH 8.8 2.5 mL

9. 10% SDS 100µL 10% APS 100µL TEMED 4µL Agarose Sealing Mixture: Solution Component Amount 1X TAE Buffer 50 Ml Agarose 0.5 g 1% Bromophenol Blue 100 µL SDS-PAGE Running Buffer (10x): Solution Component Amount (4L) Glycine 576g Tris base 121.1g SDS 40g ddH2O top up to 4L APPLICATIONS A large and growing application of 2-D electrophoresis is within the field of proteomics. The analysis involves the systematic separation, identification, and quantitation of many proteins simultaneously from a single sample. Two-dimensional electrophoresis is used in this field due to its unparalleled ability to separate thousands of proteins simultaneously. The technique is also unique in its ability to detect post- and co-translational modifications, which cannot be predicted from the genome sequence. Applications of 2-D electrophoresis include proteome analysis, cell differentiation, detection of disease markers, therapy monitoring, drug discovery, cancer research, purity checks, and micro-scale protein purification.

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