8 SDS-PAGE, ELISA, MALDI-TOF

• SDS-PAGE is a standard test that is used to determine the charged molecules, mainly proteins and nucleic acids

  • Widely used in biochemistry, forensics, genetics, and molecular biology
  • Technique for us to separate the bands of DNA templates, RNA, mRNA based on their fragments
• Laemmli system of SDS-PAGE was first introduced in 1970s
• Principle of SDS-PAGE

  • Separates protein in an electric field
  • Migrates through a liquid or semisolid medium when subjected to an electric field from anode to cathode terminal
  • Molecules flow at different rates, depending on the molecular size of proteins
• SDS-PAGE has almost the same principle with ordinary electrophoresis

  DIFFERENCE:

  • In ordinary polyacrylamide gel electrophoresis, there are factors which can affect the migration of proteins or DNA.
  • Factors include size, structure and the charge.
  • In SDS-PAGE, proteins and DNA are separated solely on size of polypeptide length.
  • It eliminates other factors.
• SDS-coated large proteins migrates slowly through the gel matrix and small proteins migrate quickly through the matrix

  • The nearer the band is to the well, the larger the molecular size of protein
• SDS is a negatively charged detergent sodium dodecylsulfate, used to denature and linearize the proteins

  • Coated the proteins with negatively charged
• SDS causes the dissolution of disulfide bonds, eliminates folds, and structure of proteins will be linear.

  • It will bear negative charge only
  • Eliminates factors of migration in electrophoresis due to charges and structures.
• In SDS, if the protein has a linear structure and only one charge, we will be able to focus on the polypeptide length.
• Without SDS, proteins with similar sizes will migrate differently because of differences in electrophoreses brought upon by factors such as charge and structure
• For complex proteins in SDS, they will be subjected first to B-mercaptoethanol or Dithiothreitol.

  • They are reducing agents.
  • Structure is loosened before applying SDS
• Polyacrylamide is used to form a gel, a matrix of pores which allow the molecules to migrate at different rates
• Concentration of gel affects migration of proteins.

  • The higher the concentration, the smaller the pores
• Polyacrylamide gel

  • The size of pores is determined by the concentration of acrylamide
  • The higher the concentration, the smaller the size of pores
  • Discontinuous SDS-PAGE consists of two different gels
• Polyacrylamide is used for gel in SDS-PAGE because:

  • It is chemically inert
  • Electrically neutral
  • Hydrophilic
  • Transparent for optical detection
• Discontinuous SDS-PAGE gels

  • Stacking gel (top gel)
  • 6.8 pH
  • 4% acrylamide
  • Larger pores, lower ionic strength
  • Separating gel (bottom gel)
  • 8.8 pH
  • Range from 5-15% of acrylamide
  • Smaller pores, higher ionic strength
• Protein bands in SDS-PAGE is visualized under UV light
• Coomassie blue is a traditional method requires staining followed by destaining to remove background gel staining

  • Most common and least sensitive
  • Detection limit: ~100 ng of protein
• Silver stain is the most sensitive test (in staining)

  • Detection limit: 0.1-1.0 ng of protein
• SDS-PAGE applications

  • Determine purity of protein samples
  • Determine molecular weight of protein
  • Identifying disulfide bonds between protein
  • Quantifying proteins
  • Blotting applications
• In SDS-PAGE, molecular weight is determined by comparing the results with a standard curve of relative mobility of standard proteins
• Enzyme-linked immunosorbent assay is first described by Eva Engvall and Peter Perlmann in 1971
• ELISA is commonly used to measure antibodies, antigens, proteins, and glycoproteins in biological samples

  • Used in the diagnosis of HIV infection, pregnancy tests and measurement of cytokines or soluble receptors in cell supernatant or serum
• ELISA assays are generally carried out in 96 well plates, allowing multiple samples to be measured in a single experiment.

  • These plates need to be special absorbent plates to ensure the antibody or antigen sticks to the surface
  • e.g., NUNC Immuno plates
• Direct ELISA uses a plate-based immunosorbent assay intended for the detection and quantification of a specific analyte from within a complex biological sample

  • e.g., antigens, antibodies, proteins, hormones, peptides, etc.

  WHEN TO USE:

  • assessing antibody affinity and specificity.
  • Investigating blocking/inhibitory interactions
• Direct ELISA

  
• Indirect ELISA is similar to direct ELISA in that an antigen is immobilized on a plate, but it includes an additional amplification detection step

  • A technique that uses a two-step process for detection

  WHEN TO USE:

  • measuring endogenous antibodies
• Indirect ELISA

  
• Sandwich ELISA is the most common type; It uses two antibodies: a capture antibody and a detection antibody

  • Antigen is bound between antibodies.

  WHEN TO USE:

  • determining analyte concentration in a biological sample
• In sandwich ELISA,

  • Capture Ab is coated on a microplate and a sample is added. The protein of interest binds and is immobilized on the plate.
  • A conjugated detection antibody (the second Ab) is then added and binds to the additional epitope on the target protein.
  • A substrate is added and produces a signal that is proportional to the amount of analyte present in the sample
• Sandwich ELISA

  
• Competitive ELISA is commonly used for small molecules, when the protein of interest is too small to efficiently sandwich with two antibodies.

  • Technique used for the estimation of antibodies present in a specimen, such as serum.
  • Principle: two specific antibodies, one conjugated with enzyme and the other present in test serum, are used
• In Competitive ELISA, instead of a conjugated detection antibody, a conjugated antigen is used to compete for binding with antigen present in sample.

  • The more antigen is present in the sample, the less conjugate Ag will bind to the capture antibody.
  • Substrate is added and the signal produced is inversely proportional to the amount of protein present in sample
• Competitive ELISA

  
• There are many different immunoassay platforms available to measure protein levels in biological fluids.

  • ELISAs are preferred in many cases due to their sensitivity, specificity, accuracy, and ability to tolerate harsh buffer or pretreatments.
• ELISAs tend to be the most sensitive immunoassays due to the binding characteristics of the antibodies and the amplification or different read-out systems used.
• Matrix-assisted Laser Desorption/Ionization Time of Flight is a powerful analytical mass spectrophotometry technique that specializes in identification of microorganisms for medical diagnosis

  • It measures the mass of molecules from a sample that has been embedded in a matrix by using a laser to ablate and desorb the molecules with minimal fragmentation
• In MALDI-TOF, resultant mass spectrum is being produced from the pattern of detected MC or MZ (mass-to-charge) ratio.

  • Its uniqueness can be leveraged for identification purposes when a comparison reference spectrum is now available
• MALDI is a soft ionization that involves a laser striking a matrix of small molecules to make the analyte molecules into the gas phase without fragmenting or decomposing them
• In MALDI-TOF, we start by ionizing our particles.

  • Some molecules are too large and decompose when heated and traditional techniques will fragment or destroy your macromolecules.
  • So MALDI is appropriate to analyze biomolecules such as peptides, lipids, saccharides or other organic macromolecules.
• During the ablation process, the gas molecules are usually ionized by being protonated or deprotonated with the nearby matrix molecules.