Electrophoresis Techniques and Applications PDF

Electrophoresis (phoresis: Greek, “being carried”) Electrophoresis - schematic In electrophoresis, charged species are propelled through a static, liquid medium by an electrical field Negatively charged species migrate...

Electrophoresis (phoresis: Greek, “being carried”) Electrophoresis - schematic In electrophoresis, charged species are propelled through a static, liquid medium by an electrical field Negatively charged species migrate from the cathode (- charge) towards the anode (+ charge) Positively charged species do the opposite Proteins can be given a net charge by adjusting the pH (e.g., high pH to give species –ve charge), or by binding anionic lipids/surfactants A gel medium can provide additional resistance Electrophoresis: theory  dV E =− E is the electric potential dx ion in E field: F = q E =zeE (Coulomb’s law) F = force q = charge on a species z = valence of ion (+2 for Mg+2, etc.) e = elementary charge (1.6 × 10-19 Coulomb) In electrophoresis, current is carried by both cations and anions. + + E = 0: protein anion surrounded by - “cloud” of shielding cations + ++ + + + + E > 0: anions and cations pulled + in opposite directions - +  Tiselius: the founder of protein electrophoresis J Exp Med. 1939 Jan 1; 69(1): 119–131. Arne Tiselius 1902-1971 Uppsala University, Sweden Nobel Prize, Chemistry, 1948 “for his research on electrophoresis... especially for his discoveries concerning the complex nature of the serum proteins” Tiselius electrophoresis apparatus Protein electrophoresis was originally performed in the liquid phase A central protein sample migrated through buffer towards either the anode or cathode Protein was then detected by refractive index changes as it moved past the detector  Electrophoresis profile of serum proteins “The method has been of importance in the problem of fractionating blood serum. Among the first results obtained by the new apparatus was that serum gives a number of relatively distinct components: albumin, , , and  globulin. It was subsequently found that further subdivision of some of these components could be made.” (Tiselius, 1948) Note that the electropherogram resembles a chromatogram The peaks are quite broad due to diffusion and convective mixing (the electric current heats the buffer) Bienvenu et al., Clin. Chem. 44: 599-605, 1998.  Gels While electrophoresis can be run in solution, modern experiments almost always use gels as a medium Gels are loose mesh-works of a polymer (chemical or a long chain polysaccharide) The spaces between the polymer are (by design) larger than a macromolecule, and filled with buffer The molecule travels through the spaces, but the gel offers resistance to movement The gel also reduces diffusion and convective mixing, improving resolution It can also act a solid support that holds the protein in place once electrophoresis stops (e.g., for band removal) This enables later staining, imaging, western blotting, or mass spectrometry  Agarose (agar) D-Gal L-Gal repeat Agarose is a polysaccharide made naturally by red algae (red seaweed) It is built as a 4)-D-Galactose(1➝4)L-Galactose(1➝ repeat Agar is used extensively in the food industry where a glutinous texture is needed – e.g. gummy bears  Agarose gels Agarose melts at high temperature At room temperature individual chains re-associate with on another lengthwise Multiple chains aggregate to form bundles Individual bundles will stick together at their overhanging ends This makes a solid mesh with large pores running throughout it Pores are on the order of 100 – 500 nm for a 1 % weight/vol agarose gel Agarose is used extensively in chromatography and DNA electrophoresis Polyacrylamide gels acrylamide Polyacrylamide gels are extensively used in chromatography (beads) and electrophoresis They are formed by the polymerization of acrylamide in the presence of small amounts of a bifunctional crosslinker, usually methylene-bis-acrylamide. The co-polymerization produces a mesh-like network of acrylamide chains with interconnections formed from the bis- acrylamide Polyacrylamide is the main gel material for protein gels Also used for DNA gels with increased resolution vs agarose (e.g. DNA sequencing) Neurotoxin Note: composition of polyacrylamide gels T = total % concentration (weight/volume) of monomer (acrylamide plus crosslinker) in the gel C = % of the total monomer represented by the crosslinker (not w/v) For example, an 8%, 19:1 (acrylamide/ bis-acrylamide) gel has T = 8% and C = 5% So 1/20th of 8 % (i.e. 0.4 % w/v) is crosslinker, the remaining 19/20 (i.e. 7.6 % w/v) is monomer  Polymerization of polyacrylamide Polymerization of polyacrylamide proceeds via a free-radical mechanism. Phase 1: initiation Requires a chemical to initiate the production of free oxygen radicals Ammonium persulfate (APS) ((NH4)2S2O8) + TEMED is added for this job APS TEMED APS serves as a source of free radicals TEMED (a tertiary aliphatic amine N,N,N',N'- tetramethylethylenediamine) accelerates their production Polymerization of polyacrylamide Phase 2: Propagation Olefin polymerization (free-radical chain reaction) RO + H2C CH2 ROCH2 CH + H2C CH2 ROCH2 CH2 CH2 CH2 Acrylamide – an amide derivative of ethene Polymerization of acrylamide C O C O C O C O C O C O NH2 NH2 NH2 NH2 NH2 NH2  Polymerization of acrylamide/ bis-acrylamide mix H2C CH C O C O C O C O C O C O C O NH2 NH2 NH NH2 NH2 NH NH2 CH2 CH2 H2C CH NH NH C O C O C O NH bis-acrylamide – two acrylamides, CH2 linked through a C O C O C O C O C O C O NH methylene group NH2 NH2 NH2 NH2 NH2 NH2 C O H2C CH Polyacrylamide Gel Electrophoresis (PAGE) apparatus Samples will be added to these wells Interface is the ”start line” for the protein race An example of an apparatus See Lab video for Labs 3 and 4 on CourseLink PAGE Note – only the gel connects the top buffer reservoir to the bottom buffer reservoir All current has to flow through the gel An analytical gel will be thin ~ 0.75 mm The sample wells are square gaps in the stacking gel where the sample can be introduced They are created by inserting a plastic “comb” before the stacking gel polymerizes The Stacking Gel  SDS + DTT = linearized protein Dithiothreitol (DTT) (also BME) is a reducing agent that will reduce any DTT disulfide bonds that otherwise prevent linearization SDS Sodium Dodecyl Sulfate (SDS) is a strongly denaturing, anionic detergent At high temperature, SDS will A protein will bind about 1.4 times its penetrate a protein, and the aliphatic weight in SDS tail will interact with non-polar surfaces This will stabilize the protein in an extended, denatured form, uniformly coated in negatively charged groups SDS coated proteins migration depends only on their MW  Protein standards SDS PAGE gels are typically run with at least one lane occupied by a protein standard This is a set of proteins of known molecular weight to which other proteins can be compared to Each band has a similar mass of protein for easy detection Typically, you would buy this; commercial standards are often pre-stained with each lane a different colour to allow easy identification You can plot the MW vs mobility and estimate the MW for an unknown protein Note that mobility is proportional to ~log MW (like gel filtration) https://www.froggabio.com/bluelf-group.html SDS-PAGE is routinely used to monitor the steps in a protein purification. SDS-PAGE gels (Laemmli, 1970) SDS coating means all proteins have similar charge/mass ratio, and are linear mobility is dependent only on mass kDa Soluble FT W1 W2 E 100- 70- 55- 40- 35- 25- 15- 10- Purification of ClpP protease; 12% gel Data from Monica Goncalves & Angelina Kim, Vahidi Lab Coomassie brilliant blue 250 Same dye as used in Bradford staining Binds hydrophobic and basic regions Staining requires an acidic environment Normally methanol + acetic acid, and the gel is heated Destaining is needed to get rid of unbound stain Typical loading is 10 mg protein (total) per lane Can detect approximately 50 ng protein band Silver staining Uses silver as the staining agent Ag+ interacts with amino acid side chains Strongest interactions with acidic (Asp and Glu), imidazole (His), sulfhydryls (Cys), and amines (Lys) Ag+ reduced to metallic Ag by incubating with reducing agent Greatest sensitivity: ~1 ng protein per band (50 x more sensitive than Coomassie) Will also stain polysaccharides Silver staining is sensitive to artifacts and contaminants, so staining can take some optimization Low compatibility for downstream mass spectrometry work. Fluorescent stains (e.g., SYPRO) Broad linear quantitation range Dyes interact with the SDS This gives consistent staining High sensitivity: Can detect 10 ng protein per band in an hour exposure Visualized using a transilluminator Reversible stain; good for downstream mass spectrometry workflows SDS-PAGE with universal staining: summary Goal: Resolve all proteins in a complex mixture, according to subunit MW. Applications: Monitoring steps of a protein purification Detecting changes in protein expression (but only if proteins of interest are sufficiently abundant to detect by universal staining) Major application of SDS-PAGE is as first step in immunoblotting, for sensitive detection of specific proteins – to be discussed. Strengths: Generally applicable, fairly cheap Good (although limited) resolution. “Workhorse” technique in any protein science lab. Weaknesses: Limited resolution, compared to (e.g.) 2-D gels, HPLC. Proteins expressed at low levels are not visible. “Western” immunoblotting Nitrocellulose is a porous, paper-like material that binds proteins tightly The nitrocellulose can also be cut into strips, each covering the expected MW of a different target protein These can then be blotted with different antibodies (e.g. an experiment and a loading control) Secondary antibodies coupled to chromogenic/light reactions highlight the bands where the target protein was present https://www.youtube.com/watch?v=VgAuZ6dBOfs “Wet” Transfer, not “semi-dry” like in our lab Specific detection: the power of western blotting 225 150 102 76 52 38 31 24 17 Ponceau Red stain 12 KIT antigen in human tumour cells (Jon. Fletcher lab, Harvard Med. School) Cells have many different proteins. No indications as to how large/how much of any individual protein  Specific detection: the power of western blotting 225 150 KIT: 145,160 102 76 52 38 31 24 17 Western blot; anti-KIT 12 KIT antigen in human tumour cells (Jon. Fletcher lab, Harvard Med. School) The Western blot allows us to visualize only a single target protein amidst the full proteome  Analysis of tissue-specific protein expression Western blotting of extracts of various mouse tissues revealed that PREL1 is widely expressed and highly enriched in tissues of haematopoietic origin. Note: Tubulin detection is used as loading control. Tubulin content does not vary much between cells, so the amount of tubulin reveals whether same amount of cellular material was loaded each time Jenzora et al., PREL1 provides a link from Ras signalling to the actin cytoskeleton via Ena/VASP proteins, FEBS Lett. 580: 455-463, 2006. Knockdown of ClpP reduces growth of AML cell lines with high ClpP expression Vahidi (U of Guelph) and Schimmer (UHN) lab – unpublished data Applying western blotting to study cell signaling: phospho-specific Abs Phosphorylation at specific residues is a critical modification in signalling to modify protein interactions or alter enzyme activities Antibodies can be raised which are both sequence-specific, and phosphorylation state-specific antibodies (PSSAs) Most PSSAs are produced by immunization of animals (usually rabbits or goats) with a synthetic phosphorylated peptide. During purification, one first removes those antibodies in the antisera that bind to the dephosphorylated antigen by using a dephospho-peptide affinity column This is followed by positive selection of the flow-through on a phospho-peptide column Alternatively, make and confirm a monoclonal antibody Mandell, Phosphorylation state-specific antibodies, Am. J. Pathol. 163: 1687-1698, 2003. Using Western blotting with PSSAs to study protein phosphorylation: inhibition of STAT5 activation by a candidate cancer drug. drug (M) 0 0.1 0.3 0.5 1 5 Andraos et al., Cancer Discovery 2: 512-523, 2012 SET-2 human leukemia cells were treated with increasing concentrations of drug (BBT594, Novartis) for 1 hour and then extracted for detection of phospho-STAT5 by Western blotting. Western blotting: summary Principle: Combine the separation power of SDS-PAGE with the sensitivity and specificity of antibody-mediated detection. Applications: Measuring expression of specific proteins under specific conditions; especially, studies of biological development, regulation, responses to stress, etc. Strengths: Highly sensitive and specific. Can be extended to studies of post-translational modifications (PTMs), e.g. by using phospho-specific antibodies. Weaknesses: Applicable only if antibodies to protein of interest are available. Susceptible to cross-reactivity/ artefacts. At best, semi-quantitative; measures changes but not absolute levels. Isoelectric focusing (IEF) High-resolution technique for separation of proteins by their isoelectric point It can separate minor variants (e.g., isozymes, post- translational modifications) that differ in pI IEF can be scaled for small-scale protein preparation Isoelectric focusing IEF gels have a stable pH gradient along their length The highest pH is oriented towards the cathode (electron source) Proteins in a region of the gel that is above their pI will have a net negative charge and migrate towards the anode Similarly, proteins at a pH below their pI will have a positive charge and migrate towards the cathode Isoelectric focusing As the pI 5 protein moves into more acidic zones, they will gain protons Eventually it will have a net charge of zero, and no longer migrate The protein will become net zero charged where its pI matches the local pH Each protein in the sample migrates to its pI, no matter where it starts, and then stays there Note if it starts to diffuse away, it will acquire charge and migrate back (hence focusing) “Since different proteins have, as a rule, different isoelectric points, the idea suggests itself of separating different components of a protein mixture by concentrating them at their characteristic isoelectric points.” (Kolin) each protein focuses at its pI Creating the IEF gel Immobilines are buffer derivatives of acrylamide Immobilines of different pKa’s can be made by picking appropriate buffer chemicals The immobilines are added to a conventional low % (e.g. 4%) acrylamide gel reaction mix, then poured in a gradient (generally with several immobilines) The ratio of immobilines in a region of a gel determines the local pH Commercial IPG strips: easy and convenient IEF Commercial immobilized pH gradients (IPGs) are supplied as dried “gel strips”; they are rehydrated in the sample protein solution, prior to use. Rehydration loading takes advantage of the fact that, in IEF, proteins can initially be distributed throughout the focusing medium. High protein loads (mg of total protein) are possible with rehydration loading.  BIORAD PROTEAN i12 IEF System Gel Side Up Assembly https://www.youtube.com/watch?v=nIXidk_DWpE Separation of post translational modification variants by IEF EPO sialylation sialic acid: 9 carbon sugar with a negative charge Purified recombinant human erythropoietin (10 μg) was applied to Novex® Pre-Cast IEF gel, pH 3-7 (Invitrogen). Isoforms of rhEPO were separated at 5 mA for 2 h and focused at 500 V for 30 min; Coomassie Blue staining The gel reveals at least 8 isoforms of EPO, each differing by the number of sialic acid modifications Son et al., Enhanced sialylation of recombinant human erythropoietin in Chinese hamster ovary cells by combinatorial engineering of selected genes, Glycobiology 21: 1019-1028, 2011. Two-Dimensional Electrophoresis IEF and SDS PAGE separate proteins by two different physical properties (pI and MW) The two methods can be combined in a single gel to get two dimensions of separation Generally, IEF is run first 2D gels allow separations of hundreds of proteins into distinct peaks (e.g. all proteins in a given cell type) Individual “spots” can then be identified by mass spectrometry  Two-dimensional IEF/SDS-PAGE (O’Farrell, 1975) “Proteomics was built around the two-dimensional gel.” Garfin, D.E., Trends Anal. Chem. 22: 263-272, 2003. 1st dimension 2nd dimension 2-D gel IEF SDS-PAGE typical gel: 20 cm x 20 cm (large) limit of detection: 1 ng spot; detect 1,000 spots from 200 g protein sample (107 cells). Issaq and Veenstra, Two-dimensional polyacrylamide gel electrophoresis: advances and perspectives, Biotechniques 44: 697-698, 2008. https://www.youtube.com/watch?v=7R_R6mbqvFk Detecting changes in gene expression: Comparing 2-D gel patterns: 1990: The hard way – by eye. Untreated E. coli cells Cells treated with menadione Greenberg et al., Positive control of a global antioxidant defense regulon activated by superoxide-generating agents in Escherichia coli, PNAS USA 87: 6181-6185, 1990. Mass spec. proteomics: automated identification of proteins from 2D gels Lovri, J., Introducing Proteomics, Wiley, 2011 p. 50 Spot Picker: spots protein samples onto MALDI MS targets for subsequent analysis by mass spectrometry (GE Life Sciences) Detecting changes in gene expression: Comparing 2-D gel patterns: DIGE (differential in-gel electrophoresis) Protein extract 1 Protein extract 2 Label with red fluor. dye Label with green fluor. dye Combine samples and run on a single 2-D gel Scan gel with excitation at specific wavelengths. GE Amersham Typhoon Molecular Imager Red, green, and blue excitation wavelengths and a choice of emission filters enable imaging of an extensive variety of fluorophores. Digitally overlay the red and green images. Application of DIGE technology: example: Romano et al., Regulation of iron transport related genes by boron in the marine bacterium Marinobacter algicola DG893, Metallomics 5: 1025-1030, 2013. Bacterial cultures were grown +/- boron (0.4 mM boric acid). Bacterial pellets were sent for 2-DIGE and mass spectrometry analysis. Total protein was extracted, labeled with Cy3, Cy4 and Cy5 dyes and subjected to IEF/SDS-PAGE. Gel scanning was carried out … Scanned images were analyzed by Image Quant software. Protein spots of interest that were differentially expressed in boron-treated versus control samples were picked and subjected to in-gel trypsin digestion, peptide extraction, and desalting prior to MALDI-TOF/MS-MS. Peptide fingerprints and partial amino acid sequence information were used for protein identification in the NCBI databases. Proteomic profile of M. algicola. Green spots represent proteins that are down-regulated in the presence of boron; red spots, proteins up-regulated; yellow spots, proteins whose expression level does not change. The 108 spots picked for quantification are circled and numbered. IEF; 2D-gels; DIGE: summary Principle: Combine IEF with SDS-PAGE – orthogonal separations – to achieve very high resolution of a complex mixture of proteins. Applications: Proteomics. Analysis of changes in protein expression. Strengths: high resolution. Weaknesses: One sample at a time; not a high-throughput technique. Must be combined with other techniques to identify proteins.