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- Proteins in the sample are coated with a detergent to denature(unfold) them and give them a negative charge
- We need linear proteins to move through gel
- Preform gel electrophoresis
- Once complete, move proteins onto a membrane
- Take completed gel and membrane and sandwich them between filter paper
-Negatively charged proteins will be drawn to the positive end
- The membrane is between positive end of chamber and the gel, so the proteins will be drawn onto the membrane
- They’ll be “blotted” onto the membrane
- This membrane is an identical copy of the gel
- Why the membrane?
- Gel is too fragile for next steps
- Antibodies could become stuck in the gel and give false results
- Once proteins have moved onto the membrane, we add antibodies to locate proteins we want to see
- The antibodies act like a flag on the target protein
- Once the membrane copies the gel, it’s moved to a chamber and exposed to primary antibodies
- This primary antibody specifically attaches to the protein we’re looking for
- Once attached, the membrane is washed so extra antibodies don’t  stick around
- A secondary antibody is then added to the membrane
- This secondary antibody attaches to the primary antibody, not the protein
- Contains a reporter enzyme that will produce light or color
- The reporter enzyme allows us to locate the proteins
- By adding the antibodies, we only see the proteins we’re looking for on thr funal membrane

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- Also known at lmmunoblot
- It’s a form of gel electrophoresis to detect specific proteins
- Detect protein of interest
- Size of proteins
- Amount of protein expressed
- We’re using a charged solution to draw proteins through a gel
- Proteins are sorted by mass and ability to bind to antibodies
Western Blot Antibodies:
- Antibodies are Y-shaped proteins that bind to a specific target (antigen)
- Western Blotting uses antigens to detect certain proteins out of a large group

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ELISA - Diluting Samples & Interpreting Results

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ELISA - Diluting Samples & Interpreting Results

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Diluting Samples:
- Why diluting the samples is necessary when using the ELISA Test!
- Can protect against false positives
- Can also show disease progression
Interpreting Results:
- Look for color change
- Amount of color change can be used to signify rate of disease progression
- More marker detected = More color change

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Step 1: Antibody Coating
- Specific capture antibody is immobilized on high protein binding plates
Step 2: Protein Capture
- Samples and standard dilutions are added to wells and will be captured by bound antibodies
Step 3: Detection Antibody
- Specific detection antibody is added to enable detection of captured protein
- If protein is absent, this antibody will attach to nothing
Step 4: Detection Conjugate(enzyme)
- Binds to detection antibody (like a signal)
Step 5: Substrate Added
- Changes color to analyze results

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- Enzyme Linked Immunosorbent Assay
- Measures antibodies, antigens, proteins, and glycoproteins in a biological sample
- Diagnosis, pregnancy tests, test for contamination, allergens, and disease progression

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- Clustered Regularly Interspaced Short Palindromic Repeats
- First discovered in E. coli and also found in archaea
- CRISPRs serve as a part of bacterial immune system
- It refers to the various systems that can be programmed to target specific stretches of genetic code
- This is so it can edit DNA in precise locations
- An enzyme called Cas9(produced by the CRISPR system) will bind to the matching DNA using a "spacer"
- Spacer: Short RNA sequences that guide the CRISPR system to target DNA
- Cas9 will cut the DNA, shutting the targeted gene off
- Research suggests CRISPR-Cas9 can be used to target and modify "typos" (aka mutations) in the human genome
- Ex: Cpf1
PROS
- Scientists can Accelerate Research
- Rapid Diagnostic
- Correct Mutations
CONS
- Ethics(how far is too far?)
- Potentially wiping out species, creating bacterial resistance
- Mutations at cut site or other places in genome

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- PCR lets us take a small section of DNA and make hundreds of copies
- This small section can be amplified and examined through many processes
- For example, gel electrophoresis
- Commonly used in forensics

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Step 1:
- Denaturation
- Heat the strands of DNA to break(denature) them apart into two separate strands
- This gives room to replicate the desired gene
Step 2:
- Annealing
- Cool DNA so primers can bind
- Primers bind to areas of the DNA that we want to amplify
- Two primers needed for each side of DNA
Step 3:
- Extension
- Heat up DNA to use taq polymerase
- Taq polymerase adds nucleotides to extend the primers and replicate DNA
- This enzyme is adapted to work in high temps
20x-30x:
- These 3 steps are repeated many, many times
- DNA made from a previous cycle is used as a template
- This ensures that the desired DNA is the only thing getting replicated

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- Polymerase Chain Reaction
- Used to make many copies of DNA region to study
- 3 Main Steps
- Denaturation
- Annealing
- Extension

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- Each line on the gel is a band of DNA
- Thicker bands mean there was a lot of DNA in that specific band
- DNA samples that have similar banding patterns will be more closely related
- Closely related organisms have similar DNA, therefore their banding patterns will be similar

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- Separates DNA by size
- Shorter DNA travels further through the gel
- Utilizes Agarose
- Agarose is very porous
- Needs a buffer
- This allows the electric current to travel through the gel
- Uses
- Crime Scenes
- Paternal/Maternal Testing
- Comparing Species
- Identifying/Observing Certain Diseases
- When separating DNA, it all has the same charge
- DNA will move prom negative to positive ends
- DNA Ladder
- Known DNA sizes that are used as a standard to compare other samples

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- Exocytosis is another form of active transport
- Cell expels/releases/removes material out of the cell
- This can include releasing neurotransmitters or proteins

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1.) Phagocytosis
- Used by amoebas to engulf a substance
2.) Receptor Endocytosis
- Special receptor proteins capture a specific target molecule
3.) Pinocytosis
- Intake of liquid

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-Movement from low to high concentration
- Moves against the gradient
-Requires Energy
-ATP
- ATP energizes the protein channel to transport molecule against the concentration gradient
- Endocytosis (3 types)
- A type of active transport where the substance fuses with the cell membrane and brings the substance inside the cell in a vesicle

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- Molecules move from high to low concentration
- Moves with the concentration gradient
- Simple Diffusion
- Very small non polar molecules can pass through the phospholipid with no help or energy
- Molecules move from high to low concentration
- Moves with the concentration gradient
- Facilitated Diffusion
- Uses a transport protein because molecule may be too big or polar
- Does not require energy
- May require a stimulus(usually a ion)

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- Maintains homeostasis by controlling what goes in and what goes out
- Phospholipid bilayer
- Heads of lipids: Hydrophilic; polar
- Tails of lipids: Hydrophobic; nonpolar