Techniques used to show gene expression

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  • Techniques used to show gene expre**ion
    • Protein
      • Western blotting
        • 3. Washing - wash buffer composed of buffered saline solution plus small volume of detergent
        • 2. Primary antibody, add 05-5mg/ml diluted in PBS or TBST wash buffer and incubate overnight
        • 4. secondary antibody binds to species-specific portion of primary antibody, can belined to biotin or reporter enzyme
        • 1. Blocking - place membrane in dilute solution of protein (3-5% BSA or TBS)
        • 5. washing
        • 6. Developing - chemiluminescent/radioactive/fluorescent detected may be used depending what secondary antibody was tagged with
    • DNA
      • Southern blotting
        • 5.Hybridise to ** DNA probe (radiolabelled)- binds only complementary sequences
        • 1. Cleave target DNA to give DNA fragments
        • 2. Restriction digest, agarose gel electrophoresis to separate fragments
        • 4. Transfer to membrane
        • 3. Denature using alkali solution
    • RNA
      • Northern blotting
        • 1. Fractionated RNA immobilised onto Nylon, stain membrane with methylene blue
        • 3. Hybridise to probe
        • Allows you to look at levels of mRNA/Transcription. Can look at cell types in which gene expre**ed, size of transcript, relative abundance of transcripts
        • 2. Can be probed with DNA/ RNA, can use radiolabelled or fluorescently-labelled nucleotides

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