Polyacrylamide gel formation

Transcription

1 Part II- Protein Identification PolyAcrylamide Gel Electrophoresis (PAGE) is the best method in protein identification, MW determination, DNA sequencing, protein-protein or protein-dna interaction etc : Polyacrylamide gel formation Poly-acrylamide and bisacrylamide solution + TEMED + Ammonium persulfate (S 2 O 8 2-2SO 4-. )

2 SDS-PAGE sample preparation Protein to be analyzed + Reducing agent ( ME or DTT) + SDS [(CH 3 -(CH 2 ) 10 -CH 2 -O-SO 3- ]Na + During heating the protein would normally precipitate, but the SDS binds to the backbone and provides a negative charge to make the denatured protein soluble. SDS, like all detergents, has a hydrophobic tail and a charged polar ion. The hydrophobic tail (ie. the dodecyl part of SDS) binds to the hydrophobic backbone of the protein, and the ionic sulfate group projects out into solution making the denatured protein soluble. SDS-PAGE Gel

3 The MW of Protein Subunits Estimated by SDS- PAGE SDS-PAGE method separate denatured polypeptides. The MW of polypeptide ca be estimated by comparison to protein of known MW. To the right is the calibration plot of standard proteins which have know subunit molecular weights. Using the plot, the MW of the unknown pure protein is determined. Only pure proteins can be used for estimating subunit MW. Determining the Subunit Composition with Native MW and Subunit MW From gel filtration chromatography, one can determine the molecular weight of the native protein. From SDS-PAGE electrophoresis method, one can determine the MW of denatured subunit polypeptides. With these two informations in hand, one can determine the polymeric forms of the native protein.

4 Native Gel Electrophoresis In typical SDS-PAGE electrophoresis, the protein is denatured by heating and adding SDS. Thus, the denatured polypeptide carries negative charges in order to migrate downward along the applied electric field. However, if macromolecules carry negative charges at a set ph, say protein/dna complexes, one can determine the binding affinity through native gel electrophoresis. The method is also called EMSA (electrophoresis mobility shift assay). Other example would be agarose gel for DNA separation. ELISA: enzyme-linked immunosorbent assay Monoclonal antibody: produced from hybridoma cell. Polyclonal antibody: produced from the serum of the immunized animals.

5 Western blot to detect target protein (1) Western blot to detect target protein (2)

6 Electrofocusing for protein pi determination * ph gradient Protein ID : Experimental Approach Mass (m/z) Run 2D gel; Stain/Image Extract peptides; MALDI-TOF analyze Edman Degradation AAA Composition Immunoblot MS Excise spot; wash; digest Database search

7 MALDI : Matrix Assisted Laser Desorption Ionization TOF : Time Of Flight Mass Spectrometry 1. Sample is mixed with matrix & dried on target 4. Ions are accelerated by an electrical field to the same kinetic energy, and they drift (or fly ) down a field free flight tube where they are separated in space kv Flight tube 5. Ions strike the detector at different times, depending on the mass to charge ratio of the ion. 2. Target is introduced into high vacuum of MS High vacuum High voltage 3. Sample spot is irradiated with laser, desorbing ions into the gas phase and starting Pulsed laser the clock measuring the time of flight. Tim e 6. A data system controls all instrument parameters, acquires the signal vs. time, and permits data processing. Centrifugation Zonal ultra-centrifugation separates particles according to their sedimentation coefficients

8 Analytical ultracentrifuge analytical ultracentrifuge can be used to characterize the behavior of macromolecules in solution. Common experiments include determining the molecular weight and shape of a molecule. It is also possible to measure the association state of the sample and to characterize its homogeneity. Rotor and monochromator In an ultracentrifuge experiment, samples are loaded into specially designed centrifuge cells. These cells are spun at a speed which causes the solute molecules to sediment The distribution of solute molecules during the experiment is monitored by an optical system. The XL-A uses a highly specialized absorbance optical system. The cells are scanned across their radius and data automatically collected for subsequent analysis

9 Sedimentation velocity Sedimentation velocity is a hydrodynamic technique. It is generally used to determine shape information for a sample. However, for a single species, the diffusion and sedimentation behavior can be used to determine the molecular weight. A high angular velocity is used to cause rapid sedimentation of the solute. This results in the formation of a sharp boundary between a solute depleted region at the top of the cell and a region of uniform solute concentration for the remainder of the cell. The concentration gradient (or dc/dr) is a convenient measure of the boundary position. Electrophoresis refers to the migration of charged electrical species when dissolved, or suspended, in an electrolyte through which an electric current is passed. Cations migrate toward the negatively charged electrode (cathode) and anions are attracted toward the positively charged electrode (anode). Neutral solutes are not attracted to either electrode. Conventionally electrophoresis has been performed on layers of gel or paper. The traditional electrophoresis equipment offered a low level of automation and long analysis times. Detection of the separated bands was performed by post-separation visualisation. The analysis times were long as only relatively low voltages could be applied before excessive heat formation caused loss of separation. Capillary electrophoresis Operation of a CE system involves application of a high voltage (typically 10-30kV) across a narrow bore ( m) capillary. The capillary is filled with electrolyte solution which conducts current through the inside of the capillary. The ends of the capillary are dipped into reservoirs filled with the electrolyte. Electrodes made of an inert material such as platinum are also inserted into the electrolyte reservoirs to complete the electrical circuit. A small volume of sample is injected into one end of the capillary. The capillary passes through a detector, usually a UV absorbance detector, at the opposite end of the capillary. Application of a voltage causes movement of sample ions towards their appropriate electrode usually passing through the detector. The plot of detector response with time is generated which is termed an electropherogram

10 Transferrin dimerization These three data sets were obtained at three different loading concentrations of transferrin. All three files were fit simultaneously to a monomerdimer equilibrium model. The fit returned a kd of about 100 M for the dimerization. The relatively small and randomly distributed residuals indicate that the model provided a good fit to the data. Sedimentation equilibrium Sedimentation equilibrium is a thermodynamic technique. It can be used to rigorously determine the solution molecular weight, association state and equilibrium constant for complex formation. In sedimentation equilibrium experiments sample is centrifuged at speeds high enough to cause some sedimenting of solute particles. As solute particles tend toward the bottom of the cell they also begin to diffuse. Eventually, the two opposing forces will be in equilibrium at every point in the cell and the solute distribution across the cell will increase monotonically