Blotting Techniques

Gel Electrophoresis

  • Gel electrophoresis is integral to all blotting techniques.
  • The specific steps aren't as important to memorize.

Steps of Gel Electrophoresis

  1. Heating and Denaturing Sample:

    • The sample is broken up.

  2. Loading Sample:

    • The sample is pipetted into separate wells. Includes a marker and samples (A, B, etc.)

  3. Applying Electric Current:

    • The gel is placed in a solution, and electric current is run through it.
    • Everything runs towards the positive end.
    • DNA and RNA are negatively charged due to their phosphate backbone, so they migrate towards the positive electrode.
    • Proteins are coated with a negatively charged substance to ensure they migrate to the positive electrode as well.

  4. Migration and Separation:

    • The gel has pores, and smaller samples of protein, DNA, or RNA have more mobility and migrate faster.

    • The amount of base pairs is inversely proportional to migration speed.

      • Larger samples (e.g., 1000 base pairs) run slower than smaller samples (e.g., 300 base pairs).

    • Markers help determine the size of samples in each lane.

    • Inverse Relationship:

      • As size increases (y-axis), distance migrated decreases (x-axis).

  5. Transfer to Filter Paper:

    • The gel is transferred to a filter paper.

  6. Incubation:

    • The filter paper is incubated with an antibody or radioactive probe that binds to the DNA, RNA, or protein.

  7. Development:

    • A film of the filter paper is developed to observe how far each sample migrated.

Reading the Results

  • The readout is similar to an old-fashioned camera film development.
  • Darker bands indicate more of a sample, while lighter bands indicate less of a sample.

Blotting Techniques

  • Mnemonic: SNOW DROP
    • S - Southern blot (DNA)
    • N - Northern blot (RNA)
    • W - Western blot (Protein)

Southern Blot (DNA)

  • DNA is enzymatically cleaved prior to electrophoresis.
    • An extra step is added
  • A radiolabeled DNA probe is used to mark the sample for development.
  • Used for:
    • Detection of genes.
    • Detection of specific nucleotide sequences.
  • Clinical Application:
    • Detection of the FMR1 gene in Fragile X Syndrome.

Western Blot (Protein)

  • An antibody binds to the protein for development.
  • Helpful for:
    • Protein expression.
    • Detecting antibodies, antigens, and other types of proteins.
  • Clinical Application:
    • Confirmation of HIV diagnosis.
      • Often initally detected with an ELISA
      • Followed by Western blot to confirm due to life changing disagnosis.

Northern Blot (RNA)

  • Used to identify RNA samples.
  • Helpful for:
    • Detecting different levels of gene expression.
    • Detecting different levels of mRNA.
    • Identifying mRNA degradation.
      • High Levels of DNA + Low Levels of RNA = mRNA Degradation
    • Identifying overexpression of oncogenes or knockouts of tumor suppressor genes.
      • No mRNA may indicate a possible knockout of a tumor suppressor gene.

Southwestern Blot (DNA-binding proteins)

  • Detects DNA-binding proteins like transcription factors.
  • Relevant for assessing the presence of certain transcription factors during research.

Review

  • Blotting techniques identify proteins, DNA, or RNA present in a sample.
  • Gel electrophoresis is used to load samples into wells alongside a marker.
  • A current pulls samples towards positive electrodes.
  • Smaller samples run faster due to their ability to slip through the gel's pores.
  • After running the gel, results are transferred to filter paper and incubated with a radioactive probe or antibody.
  • Developing a film from the filter paper shows how far each sample migrated.
  • Darker bands indicate more of a sample, while lighter bands indicate less of a sample.

Step 1 Test Questions

  • Example 1:
    • Which of these samples is the largest or has the most base pairs?
    • The samples closest to the loading zone would have the most base pairs.
  • Example 2:
    • A scientist uses CRISPRCas9 to create a knockout of p53 in a cell line but isn't quite sure that they hit both copies of the tumor suppressor gene. What is a laboratory technique that they could use to determine whether or not p53 was still functional in their cell line?
    • Western blot should be used because the questions asks about a protein that has been transcribed.
      • SNOW DROP mnemonic will then help in remembering which blot technique to use.