#Initial Phenotype of Mutant Complementation TestBeta-Gal AssayProbable Mutation 1RedWhiteBlue 2RedWhite 3RedNo growthWhite 4Red White 5Red White 6Red White 7RedWhite 8RedWhiteBlue PwhiteWhiteblue

Techniques to know Additional Techniques: Northern Blot RNAse Protection Assay Immunoprecipitation and Western Blot EMSA (Electromobility Shift Assay) IHC (Immunohistochemistry) DNA microarray

Northern Blot Analysis (Detecting Levels of mRNA) Northern blots do not provide any answers to whether levels of transcripts are due to differences in transcription or post-transcriptional mechanisms.

RNAse Protection Assay Steps: 1.A radioactive cRNA probe is synthesized by in vitro transcription using cDNA, DNA-dependent RNA polymerase (from bacteriophage SP6, T7 or T3) and [ 32 P]UTP. 2.cRNA probes are hybridized with target mRNAs in solution. 3.The reaction is incubated with RNAses specific for single-stranded RNA. The probe and the target sequence are thus 'protected' from RNase digestion by forming a double-stranded hybrid. 4.Digestion products are then analyzed by gel electrophoresis

RPA methods are time-consuming but have advantages 1.At least ten-fold more sensitive than Northern Blots 2.Multiple targets can be assayed simultaneously “multiplexing” 3.Very high specificity (mismatched RNA:probe hybrids are vulnerable to digestion by RNases. 4.Detect non-abundant and rare transcripts with high degree of accuracy

Use of biotinylated probes in RPA

Genome-wide assessment of mRNA transcripts Whole process is based on hybridization of probe to target DNA GREEN: Control DNA, RED:Experimental DNA Microarray Studies (DNA microarray, DNA chip, Gene chip, Biochip)

DNA Microarray Technique

Steps in a western blot 1.Tissue Preparation 2.Gel electrophoresis 3.Transfer (nitrocellulose or PVDF membranes) 4.Blocking (skim milk) 5.Detection Detection: 1.Colorimetric detection 2.Chemiluminescence (Substrate fluoresces when exposed to reporter on 2 o antibody) 3.Radioactive detection (Radioactive proteins are exposed to X-ray film) 4.Fluorescent detection (probe is excited by light which is measured by camera)

Western Blot Mixture of protein molecules

Chemiluminescence using Horse Radish Peroxidase-Conjugated Secondary Antibodies

Detecting Phoshorylation Status of Proteins 1.Use of commercially available phospho-specific antibodies 2.Immunoprecipitate protein of interest from crude protein sample and then blot with phospho-specific antibody. Example: Article 1 (IP for Neu and blot for P-Tyr) Detecting Interaction of Proteins on a Crude level 3.Co-immunoprecipitation: IP for protein A and blot for Protein B. This tells you whether Protein A and B interact (unless the interaction is transient).

Immunohistochemistry: a technique for identifying cellular proteins by utilizing antigen-antibody interactions To better understand the localization, distribution and amounts of proteins in the cells: Tagging of Ab with colour producing dyes or to different fluorophores. Use of fluorophores is increasing due to its use in Confocal imaging which is more sensitive and allows for visualization of interacting proteins. Clinical use: To detect presence and levels of target proteins in disease which helps to determine the type of therapy to be used. Preferred technique: better signal and more sensitive

Steps: 1. Label DNA with 32 P(this is your probe) 2. Mix radioactive DNA and protein to form complex 3. Add nonspecific competitor 4. Separate complex with free probe on a native gel Electromobility Shift Assay (EMSA) (Also known as Gel Shift Assay) This technique utilizes the rationale that DNA-protein complex will have smaller mobility than that of free DNA when subjected to non-denaturing gels

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