Membrane Class 6 for cell biology and electrophoresis

cell biology

1. Membrane Chromatography, Electrodialysis and Electrophoresis Class 5 Dr. Sandeep Kale Bioprocessing Group DBT-ICT-Centre for Energy Biosciences, Institute of Chemical Technology, Mumbai of University Matunga, Mumbai-400 019, India

2. Membrane chromatography It is evolved from the bioprocessing industry’s need to overcome the mass transfer limitations associated with conventional resin-based chromatography • Transport phenomena associated with resin bead chromatography are complex Fluid stream must be dispersed through the resin bed Solute must diffuse into the pores within the beads in order to reach the available binding sites Complex pore geometry provides additional resistance to solute transport to the binding sites

3. Electrodialysis Electrodialysis is an electrochemical process whereby electrically charged particles, ions, are transported from a raw solution (retentate, diluate) into a more concentrated solution (permeate, concentrate) through ion-selective membranes by applying an electric field. When a salt solution is under the influence of an electric field, as is the case in an electrodialysis module, the charge carriers in the solution come into motion. This means that the negatively charged anions migrate towards the anode and the positively charged cations towards the cathode. This separation stage results in a concentration of electrolytes in the so-called concentrate loop and a depletion of charge carriers in the so-called diluate loop

4. Electrophoresis is the movement in a liquid of a charged particle under the influence of an electric field. One-dimensional SDS polyacrylamide gel electrophoresis (SDS PAGE) is by far the most common for protein separations Isoelectric focusing (IEF), which separates either native or denatured proteins by their isoelectric point, useful for assessing purity or complexity of a sample and is the critical first step or dimension in 2D SDS PAGE 2D SDS PAGE is a mainstay in the field of proteomics, which studies gene expression at the protein level RNA and DNA separations use both agarose for larger molecules and acrylamide for smaller-sized nucleic acid fragments. In addition to purification, isolation, and sizing of nucleic acids, on electrophoresis, also include DNA sequencing and gene analysis. Electrophoresis

5. ELECTROPHORESIS THEORY The force driving the charged molecule through the separation medium is the product of the charge ‘Q’ on the protein or nucleic macro-ion and the potential gradient ‘ E’ across the electrophoresis chamber: QE Resistance from the surrounding medium counteracts the movement To understand electrophoretic behavior of a molecule, Stokes’s law, which approximates particle movement in a free solution, defines the resistance to electrophoresis as Where, f is the resistance of the medium to electrophoresis of the molecule, r represents radius of the particle (assumed to be a sphere), v is velocity of the particle, and η is viscosity of the liquid. These two basic equations indicate that molecule charge, size, and shape, solution viscosity, and applied voltage gradient all play a role in electrophoresis

6. Typical separation conditions for proteins and DNA in acrylamide and agarose require 500 V and 200 mA . Exceptions are found in isoelectric focusing where voltages up to 5,000 V are used, and DNA sequencing where up to 2,000 V and 80 W are used PORE SIZE AND SEPARATION MATERIAL Choice of acrylamide or agarose gel depends on the application and the size the molecule and is governed in large part by the porosity of the two matrices Polyacrylamide is formed by polymerizing the acrylamide monomers and the crosslinking agent N,N- methylene bisacrylamide with the addition of the initiator ammonium persulfate and the accelerator N,N,N,N- tetramethylethylenediamine (TEMED).

7. Agarose is a galactan hydrocolloid from agar derived from marine algae. To prepare a separation gel, powdered agarose is dissolved by heat in the electrophoresis buffer and then allowed to solidify into a gel by cooling. Typical concentrations of agarose (0.75–1.5%) have very large pores that can fractionate much larger molecules than those in acrylamide . Agarose is typically used for nucleic acid separations and to a lesser extent isoelectric focusing

8. Standardization and Visualization Separated proteins are typically visualized through equilibration with a protein-binding blue dye , Coomassie blue R 250, or by silver staining Alternative techniques include prelabeling with a radioactive amino acid, and then analysis by exposure to X-ray film in a procedure called autoradiography. Nucleic acids are in general visualized with fluorescence after soaking the gel with the intercalating dye ethidium bromide, but they can also be stained with silver and with radioactive labeling and autoradiography

9. Key Applications of Protein and Nucleic acid Electrophoresis

10. PROTEIN ELECTROPHORESIS Proteins are polyampholytes composed of amino acids containing a variety of ionizable acidic and basic groups that give the protein either a positive or a negative charge, depending on the pH At pI net charge on the protein is zero, reflecting the balance between the ionized positive and negative groups on the protein. The pI also depends on the conformation of the protein and any charged groups such as phosphorus that were added as a posttranslational modification. In the presence of denaturants or reductants that break disulfide linkages, proteins unfold and expose buried charged groups, causing an altered pI . When a protein is in its fully reduced and denatured state, both size and charge separation are reasonably accurate SDS binds to a protein at a constant 1.4 g/g protein, completely masking the native charge, giving the protein (when fully denatured and reduced) a constant charged-to-mass ratio and making size dependent separation possible

11. Native Gel Electrophoresis: Under native or non denaturing conditions, the native size, number of subunits, and isoforms can be studied Factors influencing native gel electrophoresis include native charge, shape and size of the protein Ferguson plots : First the protein sample is run under varied gel concentrations (typically 5, 10, 15, and 20%) and the slope of the log relative mobility (log Rf ) versus acrylamide concentration (%T), called Kr, or the retardation coefficient, is plotted against the size of a series of standard proteins. By comparison to the standards, the approximate size of the unknown is determined.

12. Isoelectric focusing Isoelectric focusing (IEF) separates proteins based on the intrinsic charge of the protein and is performed by adding the protein sample to a gel containing a pH gradient. Chemically synthesized additives to the electrophoresis gel ( ampholytes ) generate a stable pH gradient from the basic (cathode or negative electrode) to the acidic (anode or positive electrode) end of the gel with an applied voltage. Ampholytes not only carry charge, but also are good buffers, containing a mix of acidic (carboxyl) and basic (amino) groups. Under the influence of an electric field, ampholytes migrate either as anions or cations toward the positive or negative electrode, respectively. Ampholytes gain or lose protons, and thus charge, until the isoelectric point is reached, where the molecule has a net charge of zero.

13. Commercially available ampholytes have many species with slightly different isoelectric points that produce relatively smooth transitions in the pH gradient over a variety of ranges. The sample proteins move either toward the acid or the basic end, depending on the native charge of the protein. When the protein gets to the pH that represents its isoelectric point, the protein stops migrating and is considered focused. Initially, IEF used mobile ampholytes Now possible to create a pH gradient grafted directly onto the acrylamide matrix by pouring a gradient of ionizable groups covalently attached to the acrylamide using Immobiline chemistry

14. SDS PAGE Most commonly used electrophoresis technique for protein separation is based on discontinuous SDS PAGE Discontinuous or multiphasic systems separate proteins through a gel with two sections, the stacking and resolving regions The stacking gel contains a pH 6.8 Tris-HCl buffer as well as a nonrestrictive large-pore acrylamide gel through which the sample moves. The chloride ions (called the leading ions) have an electrophoretic mobility greater than the sample proteins. Glycine ions (called the trailing ions) in the Tris-glycine electrode, or reservoir, buffer are less mobile than the sample proteins.

15. The high mobility chloride ions leave a zone of lower conductivity between themselves and the migrating protein, causing a zone with a high voltage gradient. The proteins are then forced to “stack” in the zone between the leading and trailing ions The proteins “ destack ” upon entering the resolving, or separating, gel In the Tris-HCl pH 8.8 separating gel buffer, glycine has a faster mobility and migrates past the proteins The proteins then separate according to either molecular size in a denaturing SDS gel, or molecular shape, size, and charge in a non-denaturing gel

16. To fully disassociate the subunits and denature the protein, the sample is mixed with SDS and a reductant , such as dithiothreitol , and heated to 100C. SDS binds at 1.4 g/g protein, giving a constant charge-to-mass-ratio, leading to identical charge densities for the denatured proteins. Thus, the SDS-protein complexes separate according to size, not charge. Optimal separation depends on the polyacrylamide concentration, with most proteins separated on gels containing from 5 to 15% acrylamide and 0.2 to 0.5% bisacrylamide

17. The relationship between the relative mobility and log MW is not linear over the entire separation range But by using the linear portion of the curve, the molecular weight of an unknown protein is determined by comparison with known protein standards

18. Gradient gels expands the linear range of separation over a much wider size range than single-concentration gels These are prepared by casting a gradient from high to low concentration of acrylamide . Proteins move into a constantly increasing concentration of acrylamide and smaller pore size, with the effect of sharpening the band and giving unparalleled resolution

19. 2D-SDS PAGE 2D SDS PAGE is a fundamental technology for basic and applied research in molecular biology, protein chemistry, clinical chemistry, and toxicology In 2D SDS PAGE, proteins are first separated by isoelectric point via IEF and then by size via SDS PAGE. 2D SDS PAGE simultaneously identifies the properties such as size, isoelectric point, and posttranslational modifications of thousands proteins. The separation yields a high-purity product that is amenable to mass spectroscopy analysis, providing further details about the protein structure.

20. There are many applications of 2D SDS PAGE analysis: Understanding complex interactions between expressed proteins in a cell and the environment; Identifying proteins that are immunoactive ; Toxicology testing (where exposure to a particular drug will increase or decrease the level of one or many proteins), which is a particularly critical area where gene analysis alone does not suffice; and Quality control separation of proteins in various products for both agriculture and human consumption.

21. 2D SDS PAGE is performed by first solubilizing and denaturing the protein using nonionic detergents, chaotropics and a reductant (NP40, urea, and DTT) The proteins are separated in the first dimension by focusing either in thin strips horizontally, or in vertical glass tubes and then the proteins are separated with discontinuous SDS PAGE in vertical or horizontal systems In the first dimension, the acrylamide matrix is porous to keep sieving effects to a minimum, whereas the second dimension is performed under conditions that emphasize the size separation by single concentration or pore-gradient gel electrophoresis Although still popular, sometimes buffering capacity of ampholytes exceeds that of proteins and large quantities of proteins tend to interfere with the generation of the pH gradient. Furthermore, carrier ampholytes have a tendency to drift, shifting the pH gradient during separation and contributing to uncertainty about the protein position in the final separation

22. SDS is not compatible for use in the first dimension as it is charged and a nonionic or zwitterionic detergent needs to be used. In the second dimension, an electric potential is again applied, but at a 90 degree angle from the first field. A new technology, immobilized pH gradient (IPG) gels, helped considerably to enhance the technology of 2D SDS PAGE and make it more reproducible. With IPG gels, the pH gradient is covalently immobilized in the gel matrix during the preparation of the gel itself and the pH gradient obtained is more stable Another advantage is that, large quantities of proteins can be loaded without significant disruption of the separation pattern , permitting detailed analysis of the spots subsequent to separation by techniques such as mass spectroscopy Thus the extreme resolution capable with 2D SDS PAGE permits the identification of proteins not only by their position on the map, but by analysis of co- and posttranslational modifications, including phosphorylation , glycosylation, proteolysis, and prenylation

23. Difference gel electrophoresis (DIGE) is form of  gel electrophoresis  where up to three different  protein  samples can be labeled with  fluorescent dyes  (for example Cy3, Cy5, Cy2) prior to two-dimensional electrophoresis. After the gel electrophoresis, the gel is scanned with the  excitation wavelength  of each dye one after the other. This technique is used to see changes in protein abundance It overcomes limitations in traditional 2D electrophoresis that are due to inter-gel variation

24. QPNC-PAGE QPNC-PAGE, or Quantitative Preparative Native Continuous Polyacrylamide Gel Electrophoresis: A high-resolution technique applied to separate proteins by isoelectric point. This new and inventive variant of gel electrophoresis is used by biologists to isolate active or native metalloproteins  in biological samples and to resolve properly and improperly folded metal cofactor-containing proteins in complex protein mixtures In order to obtain a fully polymerized gel for a QPNC-PAGE, the Polyacrylamide gel is polymerized for a time period of 69 hr at room temperature. As a result, the prepared gel is homogeneous, mechanically stable and free of monomers or radicals.

25. The  pore  sizes of the gel  are very large and therefore, sieving effects become minimized during the electrophoretic separations and interactions of the gel with the biomolecules can be neglected. The separated metalloproteins (e.g. metal chaperones, prions, metal transport proteins, amyloids, metalloenzymes , metallopeptides ) are not dissociated into apoproteins and metal cofactors. The bioactive structures (native or 3D conformation) of the isolated protein molecules do not undergo any significant conformational changes by using QPNC-PAGE. Quantitative amounts of highly purified metalloproteins and protein isomers are reproducibly isolated in the different PAGE fractions Fe, Cu, Zn, Ni, Mo, Pd, Co, Mn , Pt, Cr, Cd and other metal cofactors can be identified and quantified by inductively coupled plasma mass spectrometry or by graphite furnace atomic absorption spectrometry in PAGE fractions of human, plant and animal samples

26. It has importance to determine the structure-function relationships of isolated metalloproteins in brain,  blood or other clinical samples, because improperly folded metal proteins, e.g. copper chaperone for superoxide dismutase (CCS) or superoxide dismutase (SOD), present in these biomatrices may be responsible for  neurodegenerative diseases like Alzheimer’s disease or Amyotrophic Lateral Sclerosis

27. Affinity Electrophoresis Affinity electrophoresis is useful in analytical methods (qualitative and quantitative) used in biochemistry and biotechnology. The methods include the so-called mobility shift electrophoresis, charge shift electrophoresis and affinity capillary electrophoresis. The methods are based on changes in the Electrophoretic pattern of molecules (mainly macromolecules) through biospecific interaction or complex formation. The interaction or binding of a molecule, charged or uncharged, will normally change the electrophoretic properties of a molecule. Membrane proteins may be identified by a shift in mobility induced by a charged detergent.  Nucleic acids or nucleic acid fragments may be characterized by their affinity to other molecules.

28. The methods has been used for estimation of binding constants, as for instance in lectin affinity electrophoresis or characterization of molecules with specific features like glycan content or ligand binding. For enzymes and other ligand-binding proteins, one dimentional electrophoresis similar to counter electrophoresis or to "rocket immunoelectrophoresis", affinity electrophoresis may be used as an alternative quantification of the protein. Today gel electrophoresis followed by electroblotting is the preferred method for protein characterization because its ease of operation, its high sensitivity, and its low requirement for specific antibodies. In addition proteins are separated by gel electrophoresis on the basis of their apparent molecular weight, which is not accomplished by immunoelectrophoresis, but nevertheless immunoelectrophoretic methods are still useful when non-reducing conditions are needed

29. Temperature Gradient Gel Electrophoresis  ( TGGE ) & Denaturing Gradient Gel Electrophoresis (DGGE) These are the forms of electrophoresis where temperature or chemical gradient across the gel is used TGGE and DGGE are useful for analyzing nucleic acids such as DNA and RNA, and sometimes for proteins Temperature gradient gel electrophoresis At room temperature, in the presence of at least a mM of salt, the double stranded DNA form is quite stable, and two strings tightly wrapped about each other so that there are effectively two ends. DNA is a negatively charged molecule (anion) and in the presence of an electric field, will move to the positive electrode. A gel is a molecular mesh, with holes roughly the same size as the diameter of the DNA string.

30. In presence of electric field, the DNA will attempt to move through the mesh, and for a given set of conditions, the speed of movement is roughly proportional to the length of the DNA molecule — this is the basis for size dependent separation in standard electrophoresis. The temperature raises, the two strands of the DNA start to come apart; this is melting and at some high temperature, the two strands will completely separate. However, at some intermediate temperature, the two strands will be partly separated, with part of the molecule still double stranded and part single stranded This may from one end, to make a Y shaped structure with 3 ends or from both ends to make a structure with 4 ends, or in the middle to make a bubble. Thus in D/TGGE mobility of the DNA molecule through the gel decreases drastically when these partially melted structures are formed, and, most important, the exact temperature at which this occurs depends on sequence Thus D/TGGE offers a  "sequence dependent, size independent method"  for separating DNA molecules. 

31. Denaturing gradient gel electrophoresis Denaturing gradient gel electrophoresis (DGGE) with small sample of DNA (or RNA) applied to an electrophoresis gel that contains a denaturing agent. Certain denaturing gels are capable of inducing DNA to melt at various stages. As a result of this melting, the DNA spreads through the gel and can be analyzed for single components, even those as small as 200-700 base pairs. In DGGE technique the DNA is subjected to increasingly extreme denaturing conditions, the melted strands fragment completely into single strands Rather than partially melting in a continuous zipper-like manner, most fragments melt in a step-wise process

32. Discrete portions or domains of the fragment suddenly become single-stranded within a very narrow range of denaturing conditions. Because of this distinctive quality of DNA when placed in denaturing gel, it is possible to discern differences in DNA sequences or mutations of various genes Sequence differences in otherwise identical fragments often cause them to partially melt at different positions in the gradient and therefore "stop" at different positions in the gel Placing two samples side-by-side on the gel and allowing them to denature together reveals smallest differences in two samples or fragments of DNA

33. Applications Mutations in mtDNA : TGGE was utilized to determine two novel mutations in the mitochondrial genome Microbial ecology : Sequence variations (i.e. differences in GC content and distribution) between different microbial rRNAs result in different denaturation properties of these DNA molecules. Therefore, DGGE banding patterns can be used to visualize variations in microbial genetic diversity and provide a rough estimate of the richness and abundance of predominant microbial community members DGGE of functional genes (e.g. genes involved in sulfur reduction, nitrogen fixation, and ammonium oxidation) can provide information about microbial function and phylogeny simultaneously