Techniques used to show gene expression
- Created by: kpritchard16
- Created on: 20-12-18 22:03
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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
- Western blotting
- 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
- Southern blotting
- 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
- Northern blotting
- Protein
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