Tools Of Biochemistry Flashcards
Gel Electrophoresis
DNA, Protein
Size and shape
SDS page denatures and reduces
Western Blot
Shows antibody (synthetic) for specific protein (human)
- Electrophoresis
- Blot to paper
- Antibody wash
- Tag and fluoresce
Affinity Chromatography
Looks for protein through affinity to fixed antibodies
- Lyse cells, isolate proteins
- Run through fixed column with antibodies
- Elute fixed protein and fluoresce
Epitope tagging
Looking for epitope’s antibodies, to discover protein
- Attach epitope to protein
- Wash with antibodies specific for epitope
- Fluoresce
ELISA Direct
Looking for primary or secondary antibodies
- Electrophoresis
- Antibody wash
- Flouresce the antibody tag
ELISA Indirect
Looking for secondary antibody
- Electrophoresis
- Antibody wash
- Secondary antibody attachment
- Fluoresce
ELISA Use
Good for quantification of protein amount
Immunohistochemistry
Done in situ Used to see where proteins are present in tissue 1. Wash tissue with antibody 2. Antibody binds to protein (antigen) 3. Secondary antibody attachment 4. Fluoresce
Protein Microarrays
Analytical- protein levels Functional- rate of protein interaction 1. Protein immobilized on chip 2. Washed with solution containing antibodies/substrates/labels 3. Fluoresce, yellow is the mixed color
Restriction Enzymes
Isolates DNA strands at by cleaving at sites
Cuts at palindromes to leave sticky or blunt ends
Used to see DNA
1. Separate by electrophoresis and fluoresce
2. Look at strands using audio radiography
Hybridization and Probes
Detects specific nucleotide sequences
Need other techniques to actually see it
Homo (DNA/DNA) or hetero (DNA/ RNA) duplexes
Extreme conditions raise stringency, harder to hybridize
Shorter probes are harder to bind with
Restriction Length Polymorphism
Used to look at differences in cleavage sites
- Cut with enzymes
- PCR
- Southern blotting
- Gel electrophoresis
Southern Blotting
Looking for DNA through auto radiography
- Restriction enzymes
- Electrophoresis, blot
- Hybridize with labeled cDNA
- Autoradiography
Southern Blotting
Look at RNA expression
- Gel electrophoresis, blot
- Hybridize with labeled cDNA
- Visualized with UV probe over time
DNA Microarray
Measure expression of many genes at once
- Isolate mRNA
- Reverse transcribe into DNA
- Hybridize to probes in chip
- Fluoresce
Allele Specific Oligonucleotides
Detect mutations in alleles
- Prepare chip with ASO’s
- Wash with DNA solution
- Blot colors will correspond with condition
PCR
Try to amplify specific DNA sequence
- Heat and separate strands
- Cool and hybridize with primers
- Polymerase
- Repeat to isolate and amplify sequence of interest
ASO PCR
Amplify a specific allele
Add primer for allele
TAQ won’t extend if it isn’t 100% attached
Reverse Transcriptase (RT) PCR
Quantify mRNA in tissue
- Convert mRNA to cDNA with RT
- Amplify cDNA
Sanger DNA Sequencing
Urea polyacrilimide gel electrophoresis
Use ddNTP’s
Read from bottom to top, so need to interpret from top to bottom
Automated DNA Sequencing
PCR with ddNTP’s followed by capillary gel electrophoresis
Use laser detector to see sequence
Automated DNA Sequencing
PCR with ddNTP’s followed by capillary gel electrophoresis
Use laser detector to see sequence
SalI vs XhoI
Sal is GAC Xho is GAG
[Salt] and Hybridization
Higher [salt] means better hybridization
