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MODERN METHODS in BIOCHEMISTRY •PROTEIN MODIFICATION •PROTEIN CROSSLINKING •PROTEIN STAINING •ANTIBODY MODIFICATION •IMMUNOPRECIPITATION •METABOLIC LABELLING Affinity chromatography : • matrices – preparation • Coupling of Ligand • detection of reactive groups Affinity chromatography : Principle of method Affinity chromatography Mouse brain Example Affinity Chromatography Immunoprecipitation Gel filtration Solubilise & homogenise 50,000 x g supernatant eluted complexes SDS-PAGE Affinity chromatography matrice preparation & introduce spacer Affinity chromatography : Coupling of Ligand Affinity chromatography Example COMMONLY USED AGENTS 1. Cyanogen bromide • Cyanogen bromide (CNBr) is one of the most widely used linkers in affinity chromatography. It reacts with hydroxyl groups in agarose and other polysaccharide materices to produce a reactive linker. The linker can be connected to ligands or spacers which contain primary amine groups (such as proteins). • Cyanogen bromide is extremely toxic as it releases hydrogen cyanide on acidification. COMMONLY USED AGENTS 2. Bis-Expoxides (Bis-oxirane) • Bis epoxides react readily with both hydroxyl or amino-containing gels at alkaline pH. • This produces a long chain hydrophilic reactive oxirane. This can then be linked to ligands. • The long chain oxirane itself acts as a spacer. COMMONLY USED AGENTS 3. Carbonyldiimidazole • N,N'carbonyldiimidazole (CDI) is reacts with polysaccharides to form imidazole carbonate derivatives. These derivatives then react with ligands containing primary amino groups at an alkine pH to give stable carbonate derivatives. 4. Sulphonyl chloride • Reacts with hydroxyl groups on the matrix to form sulphonyl esters. These esters react with amino and thiol groups of the ligand. 5. Sodium periodate (NaIO4) • Reacts with diol groups on polysaccharide matrices to form reactive aldehydes. These react with primary amines to form Schiff's bases.The Schiff's bases can be stabilized by reduction with sodium borohydride. • Periodate is easy to use and non-toxic. The product is stable. 6. Glutaraldehyde • Activates amino or amide groups on polyacrylamide matrices or on agarose with amine spacers. These groups can then react with primary amine groups on the ligand. Tags and Fusion sequences • Many vectors are now engineered with DNA sequences encoding a specific peptide (purification tag) that, when fused to the expressed protein, may be used for one-step purification of the recombinant protein by high-affinity binding. That is molecular biologists design expression systems such that the recombinant protein can be easily purified using affinity chromatography. • The technique involves linking the gene coding for tag to that coding for the protein of choice. When the latter is expressed, the resulting tag is also produced and is linked to the desired protein. The tag is selected such that it can be readily be bound a particular affinity chromatography material. • Example 1 : 6xHis system : six histidine residues are tagged to the recombinant protein. The histidine tag and protein can be purified using a nickel-chelating resin. • Example 2 : pMal system : tags = maltose binding protein to the recombinant protein. The maltose binding protein tag will bind strongly to an amylose column. Brand Name Ligand Application Examples HiTrap Protein A Protein A IgG, IgG subclasses, fragments of IgG, IgA, antigens, immune complexes HiTrap Progein G Recombinant Protein G IgG, IgG subclasses lacking the albumin binding region HiTrap Heparin Heparin Growth factors, coagulation proteins, plasma proteins, lipases, lipoproteins, enzymes that act on nucleic acids, steroid receptors, protein synthesis factors HiTrap Chelating Iminodiacetic acid Proteins and peptides with exposed histidine groups HiTrap Blue Cibacron Blue F3GA Enzymes requiring adenyl-containing cofactors, albumin, coagulation factors, interferon Brand Name Ligand Affi-Gel Protein A Affi-Prep Protein A Protein A Applications Purification of IgG from acites, serum and culture fluid. Affi-Gel Blue Cibacron Blue F3GA Binds many nucleotide-requiring enzymes, albumin and other proteins. DEAE Affi-Gel Blue Cibacron Blue F3GA and DEAE groups. Binds albumin and serum proteins Used to purify protease-free IgG from acites, serum and cutlure fluid Affi-Gel heparin Heparin Purification of a wide variety of proteins including growth factors, coagulation factors, DNA and RNA specific enzymes, lipase, lipoproteins and proteases Affi-Prep polymyxin Polymixin Endotoxin removal Affi-Gel 501 Organomercurial Adsorbtion of sulfhydrl proteins and low mw sulfhydrls via thiol groups. Bound proteins are eluted with dilute mercaptoethanol or dithioreitol. Affi-Gel 601 Boronate Adsorbtion of cis-hyroxyl containing molecules including sugars, nucleotides and glycocpeptides. MODERN METHODS in BIOCHEMISTRY •PROTEIN MODIFICATION •PROTEIN CROSSLINKING •PROTEIN STAINING •ANTIBODY MODIFICATION •IMMUNOPRECIPITATION •METABOLIC LABELLING PROTEIN STAINING PROTEIN STAINING • Amidoblack • Commassie • Ponceau-red • Silver • Gold • Gelcode Protein Imaging Coomassie Blue Dyes - commonly used - does not interfere with subsequent protein identification - inexpensive - sensitivity well below silver and fluorescent dyes Silver stain - sensitivity 10-50 times greater than CB - ability to detect 1 ng of protein - silver diammine/silver nitrate - relatively expensive (reagents/waste disposal) - high background Fluorescent Stains and Dyes - accurately determine changes in protein expression - greater sensitivity than silver stain - DIGE - cost Silver-stained two-dimensional polyacrylamide-gel PROTEIN STAINING Amidoblack spot 5 ul protein sample on nitrocellulose (NC) - air-dry for 5 min - immerse in stain solution for 3 min - wash 2x 3 min in water wash 2x 3 min in wash solution wash 5 min with water air-dry 5 min - elute stain with 1 ml elution solution (while shaking the sample) - measure absorption at 630 nm of the eluant PROTEIN STAINING Amidoblack stain = 0.1 % amidoblack, 45% methanol, 10% acetic acid wash = 90% methanol, 2% acetic acid elution = 50% ethanol, 50% 50 mM NaOH/0.1 mM EDTA - use BSA solutions (0 to 5 mg/ml) for calibration - this protocol also works for proteins in SDS-PAGE buffer PROTEIN STAINING Coomassie Blue • 1. Immerse gel in 50% ethanol/10% acetic acid for at least 1 hr. • 2. Soak in 5% ethanol/5% acetic acid overnight or for a minimum of 2 hours. • 3. Wash in diH2O for 1 hr. • 4. Add Gel-Code Blue Stain reagent for at least 3 hrs. • • • (Pierce, #24592) 5. Wash in diH2O twice, 15 min each. 6. Rinse in diH2O for 1 hr. 7. Gels can be stored at 4°C. PROTEIN STAINING Coomassie-Blue &Amidoblack PROTEIN STAINING Ponceau Red • For Membrane after Blotting. To check blotting • Reversible: gets washed out with destaining solution • Ponceau S stock solution: 200 mg Ponceau S per 100 ml 3 % Perchloric acid • ready-to-use solution : dilute stock sol. 1:5 with 10% acetic acid • Background destaining :10 %acetic acid PROTEIN STAINING Silver PROTEIN STAINING OTHER STAINS Gold-Stain Gold-Blot Silver-Stain Sypro-Orange BioRad-Zinc Coomassie-Blue Sypro-Red MODERN METHODS in BIOCHEMISTRY •PROTEIN MODIFICATION •PROTEIN CROSSLINKING •PROTEIN STAINING •ANTIBODY MODIFICATION •IMMUNOPRECIPITATION •METABOLIC LABELLING IMMUNOPRECIPITATION IMMUNOPRECIPITATION APPLICATIONS-I Immunoprecipitation can be used for many purposes : • 1) Determination of the molecular weight and isoelectric point of immunoprecipitated proteins by one-dimensional or two-dimensional SDS-PAGE. • 2) Verification that an antigen of interest is synthesized by a specific tissue (i.e., that radiolabeled protein can be identified in tissues or cells cultured with radiolabeled precursors). • 3) Determination of whether a protein contains carbohydrate residues by evaluating whether immunoprecipitated antigen from cells cultured with radioactive monosaccharides is radiolabeled. IMMUNOPRECIPITATION APPLICATIONS - II • 4) Characterization of the type of carbohydrate present on glycoproteins - evaluate incorporation of different radiolabeled monosaccharides into immunoprecipitated protein during cell culture and test whether inhibitors of glycosylation alter the molecular weight of immunoprecipitated protein. • 5) Determination of precursor-product relationships by performing pulse-chase labeling followed by immunoprecipitation. • 6) Quantification of synthesis rates of proteins in culture by determining the quantity of immunoprecipitated, radiolabeled protein. Immunoprecipitation Mixed proteins Agarose bead conjugated to secondary antibody Add antibody Add beaded αbody = HA-VEGFR (Haemagglutinin) resuspend pellet and load on gel IP - anti HA probe - α p-VEGFR Spin Immuno-co-precipitation = shc Primary Secondary (mouse α-HA) (rabbit α-mouse) IP - anti HA probe - anti shc Primary sheep α Shc Secondary donkey α sheep VEGF IP anti HA VEGF IP anti HA Warner et al Biochem. J. (2000) 347, 501–509 Kinase assays 1. Add radioactive phosphate and substrate 2. Subject to SDS PAGE 3. Develop against film 32P 32P 32P labelled substrate Unincorporated 32P Porcine aortic endothelial cells tranfected with VEGF-R2 PI3 Kinase inhibitors Vehicle Stimulant Qi & Claessen-Welsh, Exp Cell Res 263 173-182 (2001) IMMUNOPRECIPITATION Explants cultured in the presence of [35S]met incubated w. Ab to uterine milk protein (UTMP). Ag-Ab absorbed w. Protein A-Sepharose SDS-PAGE + fluorography. Ab = proteins immunoprecipitated with rabbit antiserum to UTMP NRS = normal rabbit serum TC = total array of radiolabeled proteins present in the unabsorbed sample IMMUNOPRECIPITATION SOME PROBLEMS I • 1) crossreactivity : attention must be given to antibody cross-reactivity with other antigens (like all immunochemical procedures) • 2) Nonspecific binding : can be a problem especially if proteins that are immunologically distinct from the antigen are trapped in the pellets formed during immunoprecipitation. IMMUNOPRECIPITATION • To reduce nonspecific binding, immuno-precipitation buffers usually have – some detergent to reduce hydrophobic interactions, – a protein to block nonspecific binding sites, – and high salt to reduce ionic interactions. • In many protocols, a preclearing step is performed to remove molecules that nonspecifically bind to insoluble Protein A or Protein G. • Despite these precautions, nonspecific binding can occur. IMMUNOPRECIPITATION It is crucial, therefore, to always perform a control reaction where antibody is replaced by a non-relevant immunoglobulin (i.e, normal serum for polyclonal antibodies, control mouse ascites fluid for ascites, and isotype controls for purified mouse monoclonal antibodies). IMMUNOPRECIPITATION SOME PROBLEMS - II • 3) Proteolytic digestion : can occur when cells are lysed and contents of lysosomes are mixed with other compartments of the cell. Accordingly, most immunoprecipitation buffers contain proteinase inhibitors. • 4) Care should be taken in using immunoprecipitation as a quantitative tool to determine rates of synthesis of proteins because the rate of incorporation of radiolabel into protein will depend upon rate of synthesis of a protein as well as the rate of dilution of radiolabeled precursor by the intercellular pool of precursor. IMMUNOPRECIPITATION SOME PROBLEMS - III • 5) Protein Trapping : molecules too small or too large to be resolved by SDS-PAGE are sometimes trapped in the pellet formed by immobilized Protein A or Protein G. • These molecules, while not interfering with analysis by SDS-PAGE, can make direct quantification of radiolabeled antigen by scintillation spectrometry problematic. • Thus, immunoprecipitated protein should be quantified by densitometric analysis of autoradiographs or fluorographs. Scintillation spectrometry sometimes reveals little difference in the quantitative yield of radioactivity between an immunoprecipitation reaction and a control reaction (i.e., where antibody has been substituted with normal rabbit serum). Nonetheless, subsequent analysis by SDS-PAGE reveals precipitation of radiolabeled protein in the antibody reaction only. IMMUNOPRECIPITATION SOME PROBLEMS - IV • 6) Sensitivity : can be a problem, especially when the antigen is a minor component of the protein pool. New screen technology for low energy radioisotopes (Transcreen LE enhancing screens by Kodak) increases sensitivity greatly. Use as much protein in the immunoprecipitation reaction as possible. III. FASPS (4) Serine to glycine mutation in hPER2 of FASP person Fig 13. Mapping of the CKIε binding domain of PEFig 14. In vitro CKIe phosphorylation of wild R2 by immunoprecipitation(Toh et al, 2001). type and mutant hPER2(Toh et al, 2001). anti-MYC antibody mper2 cDNA PER2 myc PER2 CKIε rabbit reticulocyte ck1ε cDNA myc CKIε Immuno-precip itation Separation in SDS P AGE gels <Experimental scheme> -8- MODERN METHODS in BIOCHEMISTRY •PROTEIN MODIFICATION •PROTEIN CROSSLINKING •PROTEIN STAINING •ANTIBODY MODIFICATION •IMMUNOPRECIPITATION •METABOLIC LABELLING METABOLIC LABELLING • Aims: METABOLIC LABELLING • Aims : An important measurement of metabolism in metabolic engineering is in vivo metabolic flux: the rate of flow of biochemical material down a pathway. A B C D Rates expressed in units of quantity for unit time per unit tissue mass e.g. nmol.min nmol.min-1.g-1 Fw Metabolic Flux Analysis (MFA) Stoichiometric flux balance analysis Isotopic flux balance analysis : steady-state steady-state radiolabeling non-steady-state non-steady-state dynamic kinetic models stable isotope labeling steady-state All types of MFA involve algebraic representations of metabolism with numerous interacting variables. Effective handling of these interacting variables requires computer simulation models. Principles of flux determination from radiolabeling kinetics (external radiolabeled M) M* J M J The flux, J, can be determined by introducing a pulse of radiolabeled M (M*) and measuring the radioactivity in samples of purified M. The pool of intracellular radiolabeled M will change according to the following equation: dM* M* dt M J =Stephanopoulos, G.N., Aristidou, A., Nielsen, J. 1998. “Metabolic Engineering Principles and Methodologies”. Academic Press, San Diego. Radiolabeling Experiments 14C, 33P-labeled precursor Leaf disk labeling 1. Labeled metabolite of known specific activity supplied 2. Extraction of leaf tissue 3. Phase separation 4. Ion exchange chromatography 5. Thin layer chromatography or electrophoresis 6. Quantify metabolites by scintillation counting Practical concerns Is the supplied labeled compound taken up? Is it altered before uptake ? (e.g., phosphorylated compounds) Can you separate all relevant metabolites? Are the metabolites efficiently recoverable? Advantages of Radiolabeling Experiments • Very high sensitivity - can quantify turnover in small pools - only small tissue quantities are required; replication, many time points possible fore kinetic studies • Straightforward quantification (scintillation counting) - must account for quenching (pigments, solvents) - metabolites must be separable so that label in different compounds not counted together. - metabolites not labeled via intersecting pathways - need to consider the potential for labeling of multiple atoms • High time resolution - critical for rapid kinetics METABOLIC LABELLING How long should cells be labeled? • The ideal length of time to label cells depends on the protein of interest and the label that you are using. • If you want to label an unstable protein with 35S-methionine, a short labeling interval--no more than 2 hr—is best. • If you are studying a stable protein, a longer label may be preferable. • The issue is the half-life of the protein of interest relative to the half-life of the background bands. The half-life of total cellular protein is 45 to 50 hr. METABOLIC LABELLING Labeling with 32Pi is different. • the phosphate in proteins undergoes continual turn-over in most cases. Therefore, both old and newly-synthesized proteins become labeled soon after 32Pi is added to the cells. • A short labeling period with 32Pi is advantageous : labeling of RNA and DNA is less obvious than in cells labeled over-night. • For studying tyrosine protein kinases: phosphotyrosines tend to turn-over faster than the bulk of either phosphoserine or phosphothreonine and thus are preferentially labeled during brief labeling. METABOLIC LABELLING • over-night labeling period is optimal if looking at the steady-state abundance of phosphate in proteins, lipids, or RNA, • Under these conditions, the specific activity of the ATP pool in the cell and of the phosphates in macromolecules is beginning to approach that of the medium. • Additionally, the amount of label present in proteins, lipids and RNA should now reflect the amount of phosphate present, rather than the rate of turn-over of the phosphate. METABOLIC LABELLING • An issue to consider : radiation damage !!! • Can induce the stabilization of p53 and cause cell cycle arrest. • Cells labeled for a prolonged period with 32P may therefore not be growing when you harvest them. • the specific activity of the ATP pool comes to equilibrium with the phosphate in the medium by 6 hours. Therefore there is no reason to label for longer with 32Pi. METABOLIC LABELLING • Overnight labeling is best done in medium containing a reduced concentration of phosphate or methionine. 10% is often reasonable, depending on the cell line. • easily accomplished by using methionine-free or phosphatefree medium and 10% undialyzed serum (which can be assumed to contain the same concentration of methionine or phosphate as normal medium). METABOLIC LABELLING • For short term labeling (30 seconds to 5 hrs) Use : • • • • (1) medium completely lacking either phosphate or methionine, (2) serum which has been dialyzed against saline, and (3) a fairly low volume of medium; 0.75 ml for a 35 mm dish, 2 to 2.5 ml for a 50 mm dish, and 2.5 to 5 ml for a 100 mm dish. • It is a good idea to rinse the cells you are going to label with labeling medium—which lacks label—prior to adding the actual labeling medium. • Starvation doesn't help much.