Biomedical Techniques CLSB-222

Biomedical Techniques CLSB-222

• Course instructor: Dr. Ahmad Alamri, M.H.S, Ph.D
• Instructor email: [email protected]

Electrophoretic Techniques

• Electrophoresis is defined as the migration of charged molecules in an electric field
• It's a common technique in clinical laboratories for isolating and quantifying serum proteins, isoenzymes, hemoglobin, and lipoproteins
• Electrophoretic techniques are used in DNA sequencing, separation, and purification of biomolecules, and medical research
• Factors affecting electrophoresis include:
  • Net electric charge on molecules (pH and ionic strength of buffer)
  • Size and shape of molecules
  • Electric field strength
  • Nature of supporting media
  • Temperature of operation

A) Paper Electrophoresis

• A method of electrophoresis performed on filter paper
• Useful for separating small charged molecules like amino acids and small proteins
• Samples are spotted on filter paper, and a high voltage is applied which causes molecules to migrate towards either the positive or negative pole based on charge.
• Different staining methods can be used to detect separated components based on chemical identity.

B) SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

• SDS-PAGE is a widespread method for protein separation in biochemistry, forensics, genetics, and molecular biology.
• It separates proteins based on their molecular weights.
• Principle: SDS is a detergent that denatures proteins, binds to hydrophobic regions, disrupts non-covalent bonds, and gives proteins a negative net charge, so proteins migrate based on their size.

Principle (Cont'd)

• Smaller proteins migrate faster than larger ones through the gel affected by electrical field strength
• The number of SDS molecules binding is proportional to protein size, thus, proteins in the electrical field migrate towards the anode (+) and are separated by molecular weight.
• During PAGE, the rate of migration of SDS-treated proteins is determined by molecular weight.

Polyacrylamide Gel

• Formed by the co-polymerization of acrylamide and N,N'-methylene-bis-acrylamide.
• To polymerize the gel, a system comprising ammonium persulfate (initiator) and tetramethylenediamine (TEMED) is added.

Staining the Gel

• Staining the gel with a buffer containing glacial acetic acid, methanol, and Coomassie brilliant blue 250-R, visualizing bands.
• Destaining the gel with a buffer containing glacial acetic acid and methanol to remove excess stain.

C) Agarose Gel Electrophoresis

• Used for separating DNA fragments based on size using agarose gel
• Separates DNA by rate of movement through gel affected by an electrical field.
• Useful in determining presence and size of PCR products.
• DNA is negatively charged.
• In electrophoresis, DNA migrates towards positive pole (anode).
• Agarose gel is used to slow down DNA movement and thus, separate DNA according to size.

DNA Ladder Standard

• Contains DNA fragments of known sizes.
• Used to determine the sizes of unknown DNA.
• Bromophenol blue on the gel migrates at approximately the same speed as 300 bp DNA.

Staining the Gel (Cont'd)

• Ethidium bromide binds to DNA, allowing visualization under UV light
• Ethidium bromide is a mutagen, thus, gloves must be worn.

Sample Preparation

• DNA samples are mixed with 6X sample loading buffer (bromophenol blue for color, glycerol for weight)
• This allows visualization of samples during loading and increases the sample density for better placement in gel wells.
• Carefully place pipette tip over well and gently expel the sample. Avoid puncturing the gel.

Running the Gel

• Place the cover on the electrophoresis chamber. Connect the electrical leads to the power supply ensuring DNA migrates to the positive/anode.
• Bubbles should form on the electrodes in the electrophoresis chamber when the power is turned on.
• Make sure the gel is running in the correct direction (DNA to positive pole). Bromophenol blue runs in the same direction as DNA.

D) Southern Blotting

• A technique for detecting specific DNA sequences in samples.
• Steps involve:
  1. Digestion of DNA with restriction enzymes
  2. Separating DNA fragments by agarose gel electrophoresis
  3. Denaturing DNA
  4. Transferring DNA to a solid support (nylon or nitrocellulose)
  5. Hybridizing immobilized DNA to a labeled probe (DNA or RNA)
  6. Detecting bands complementary to the probe (autoradiography)
  7. Estimating band size, and relative amount of target DNA

E) Northern Blotting

• A method to detect specific RNA sequences in samples
• Techniques include RNA extraction, electrophoresis, and hybridization using labeled probes

F) Western Blotting ("Immunoblotting")

• Separate and identify proteins on a gel
• Used to determine protein molecular weight and relative amounts.
• Method for detection of specific proteins by using antibodies
• Procedure involves gel electrophoresis, transfer to membrane, and detection