1 Flashcards
Transfer to a solid support
Element two of WB
USed in research to separate and identify proteins
Western blot
Separation by size
Element one of WB
Marking target protein with antibodies
Element three of WB
Prevents antibodies from binding to membrane nonspecifically
Blocking
Confirm the identity of the protein and activity of the antibody
positive control
Null cell line used to confirm that the staining is not nonspecific
negative control
Slightly acidic with lower acrylamide concentration to make a porous gel to separate proteins poorly but allows them to form thin, sharply defined bands
Stacking gel
Basic with high polyacrylamide making the gels pores narrower
Separating gel
USes electric field oriented perpendicular to the surface of the gel causing proteins to move out of the gel and onto the membrane
Transfer
High affinity for protein and its retention abilities. Brittle and doesn’t allow the membrane to be used for reprobing
Nitrocellulose membrane
Important for minimizing background and removing unbound antibodies
Washing
Detects the protein of interest, Monoclonal or polyclonal
Primary antibody
Binds to conserved regions on the primary antibody and acts to amplify the signal, increasing detection sensitivity. Labeled with either an enzyme for colorimetric or chemiluminescent detection
Secondary antibody
A chemical reaction in which an enzyme catalyzes the oxidation of a chemical substrate, and the reaction produces light as a byproduct.
Chemiluminescent
Primary or secondary antibody labeled with fluorescent molecule excitable by light
Fluorescence detection
RNA is transcribed into complementary DNA by reverse transcriptase from total or messenger RNA. cDNA is used as a template for qPCR reaction
RT-qPCR
RT and PCR are performed separately with optimized buffers, reaction conditions, and priming strategies
Two step RT-qPCR
OFten used due to disadvantages for template of RT such as fewer purification steps to ensure more quantitative recovery, better normalization, and it avoids enrichment steps
Total RNA
Standardized nuclei extraction by sonication, followed by intranuclear enzymatic chromatin cutting and barcode ligation
RELACS step
Assay for transposable accessible chromatin with high-throughput sequencing, a method for mapping chromatin accessibility genome wide
ATAC-seq
Quantify DNA concentrations from multiple samples by analyzing fluorescent signal intensities that are proportional to the amount of amplicon after completing chip assay and sample purification
ChIP-qPCR
Starts with crosslinking of DNA protein complexes, then samples are fragmented and treated with exonuclease to trim unbound oligonucleotides. Protein-specific antibodies are used to immunoprecipitate the DNA-protein complex. DNA is extracted and sequenced
ChIP-seq basis
Better mechanical support and allows the blot to be reprobed and stored. Background is higher and requires careful washing
PVDF membrane
Enzyme used to amplify the signal in photometric assays through catalyzing the conversion of chromogenic or chemiluminescent substrates for the detection of targets
Harseradish peroxidase
Combines RT and PCR in a single tube and buffer using RT along with DNA polymerase. Utilizes specific primers
One step Rt-qPCR
More sensitivity for template of reverse transcriptase
mRNA
Stretch of thymine residues that anneal to the poly A tails of mRNA
OligodTs
Enzyme that makes DNA from RNA. Want a high thermal stability
Reverse transcriptase
- Predenaturation if RNA template has a high degree of secondary structure
- Primer extension
- cDNA synthesis makes complement DNA strand to the mRNA template
- Reaction termination prevents qPCR inhibition by active reverse transcriptase
cDNA synthesis
Barcoding method to enable high throughput ChIP_seq using common molecular biology techniques. Restriction enzyme-based labeling of chromatin in situ
RELACS
Identifying genome wide DNA binding sites for transcription factors and other proteins
ChIP-seq
DNA binding dye that drastically increases fluorescence when bound to double-stranded DNA
SYBR green
