micro lab 6
Key metrics of BLAST pt 2
% Identity: Percentage of identical matches between your sequence and database hits
what is BLAST
(Basic Local Alignment Search Tool)A tool used to compare a sequence (like DNA or protein) against a database to find similarities.
Size conversion
500bp = .5kb 1500bp= 1.5 kb
interpretation of western blot
A band at 50 kD matches the expected size, indicating the target protein is present and abundant.
what is GroEL
A chaperonin that helps proteins fold correctly and prevents aggregation.
what is a westen blot
A method to detect specific proteins in a sample.
What is gel electrophoresis?
A method to separate DNA fragments by size.
what is an SDS-PAGE
A technique to separate proteins by size.
performing a BLAST search pt 1
Access BLAST Tool: Go to the BLAST website.
Key metrics of BLAST pt 3 a.
Accession Number: Identifier of the matched sequence.
key points of a western blot pt 5
Post-Translational Modifications: Unexpected sizes may indicate modifications.
significance of findings pt 2
Potential Applications: Findings can improve stress tolerance in crops and inform medical research.
what is SodA (superoxide dismutase)
Protects cells from oxidative damage by converting harmful superoxide radicals to less harmful substances.
key points of a western blot pt 1
Protein Bands: Indicate presence; brighter bands mean more protein.
western blot analysis pt 1
Protein Extraction: Proteins are extracted from both wild-type and mutant samples.
Key functions of SDS-PAGE pt 1
Protein Separation: Based on molecular weight.
Key functions of SDS-PAGE pt 2
Purity Assessment: Single bands indicate pure proteins; multiple bands suggest contamination.
family
Related genera
Determine protein size from SDS-PAGE pt 1
Run SDS-PAGE: Load samples with a protein ladder.
steps to estimate size pt 1
Run a DNA Ladder: Include a marker with known sizes alongside your samples.
western blot analysis pt 2
SDS-PAGE: Proteins are separated by size using SDS-PAGE.
experimental design pt 2
Sample Collection: Samples are collected for Western blotting to analyze protein levels.
Key metrics of BLAST pt 3. c.
Score: Higher scores mean better alignments.
performing a BLAST search pt 4
Select Database: Choose where to search
Key metrics of BLAST pt 1
Sequence Length: Found at the top of the results (in base pairs for DNA).
Key functions of SDS-PAGE pt 3
Size Estimation: Compare band positions to a molecular weight marker.
key points of a western blot pt 3
Specificity: Use controls to confirm results.
steps to estimate size pt 2
Stain the Gel: Use a dye to visualize DNA bands under UV light.
Determine protein size from SDS-PAGE pt 2
Stain the Gel: Use a dye to visualize bands.
experimental design pt 1
Stress Treatment: Both normal (wild-type) and mutant strains are exposed to stress
performing a BLAST search pt 5
Submit Search: Click to run the search.
Key functions of SDS-PAGE pt 5
Western Blotting Preparation: Often used to transfer proteins for further detection.
steps to estimate size pt 5
estimate Sizes: Use the curve to find sizes of unknown bands.
How do you name a bacteria
genus and species
key points of a western blot pt 4
Loading Control: Ensures equal protein loading across samples.
how do you find closely related species
Look for species on branches that are close together; shorter distances mean closer relationships.
types of mutants pt 1
Loss-of-Function Mutants: These lack the target gene's function, helping to understand its role during stress.
steps to estimate size pt 3
Measure Band Position: Record how far each band traveled.
Determine protein size from SDS-PAGE pt 3
Measure Migration: Record the distance each band traveled.
key points of a western blot pt 2
Molecular Weight: Compare to markers to estimate size.
species
Most specific level; individuals that can interbreed
Key metrics of BLAST pt 3 d.
E-value: Lower values indicate more significant matches.
interpreting results of western blot pt 3
Multiple Proteins: Analyzing several proteins provides a broader view of stress management.
importance of bacterial classification
Helps in identifying and classifying bacteria, understanding relationships, and aiding research and medicine.
Phylum
Highest rank; groups with major similarities
Determine protein size from SDS-PAGE pt 4
Create a Standard Curve: Graph distances vs. known sizes.
steps to estimate size pt 4
Create a Standard Curve: Graph the known sizes against their migration distances.
interpreting results of western blot pt 1
Decreased Protein Levels: If a protein is lower in the mutant, the gene is likely important for stress response.
Key metrics of BLAST pt 3 b.
Description: Brief info about the match.
what are phylogenic trees
Diagrams showing evolutionary relationships among species.
Key metrics of BLAST pt 3
Best Matches: Results show top matches ranked by significance
what are the components of phylogenic trees
Branches, nodes, tips
Key functions of SDS-PAGE pt 4
Characterization: Analyze protein mixtures and post-translational modifications.
performing a BLAST search pt 2
Choose Program: Select the type of BLAST (e.g., nucleotide or protein).
genus
Closely related species
Determine protein size from SDS-PAGE pt 6
Convert to Daltons: 1 kD = 1,000 Da.
Determine protein size from SDS-PAGE pt 5
Estimate Sizes: Use the curve to find unknown sizes.
examples of fluorescent indicators
Fluo-3, SNARF
examples of fluorescent dyes
Fluorescein, Cy3 and Cy5:
types of mutants pt 2
Gain-of-Function Mutants: These enhance gene function to study effects of overexpression.
creating mutants in western blot
Gene Knockout/Knockdown: Mutants are created by removing or reducing specific genes using methods like CRISPR or RNA interference.
examples of fluorescent proteins
Green Fluorescent Protein (GFP),mCherry
interpreting results of western blot pt 2
Increased Protein Levels: Higher levels may indicate compensatory mechanisms due to the missing gene.
function of fluorescent indicators
Indicate changes in pH or ion concentrations.
performing a BLAST search pt 3
Input Sequence: Enter your query sequence.
western blot analysis pt 3
Transfer and Detection: Proteins are transferred to a membrane and probed with antibodies to identify stress response proteins.
significance of findings pt 1
Understanding Mechanisms: Helps clarify how specific genes influence stress responses.
function of fluorescent proteins
Used as markers in live-cell imaging.
function of fluorescent dyes
Used to label and visualize cells, proteins, or nucleic acids.
