Laboratory Techniques Bio and Chem Flashcards
In general when doing Western blotting the ____ will migrate down quickly through the gel
Shortest amino acid sequence have lower molecular weight and migrate down quickly through the gel
___ can detect and quantify proteins.
Enzyme Linked immunosorbant Assay (ELISA)
How does ELISA work?
A primary antibody (linked to a “reporter” enzyme) is added, which binds the antigen (protein). Samples are then washed to remove unbound proteins, and the reporter enzyme substrate is added. Enzyme-substrate reaction creates product that results in a quantifiable/detectable signal
__ is used to analyze mRNA expression levels
Northern Blot
__ is used to detect proteins
Western Blot
__ is used to separate proteins by molecular weight
SDS-Page
What are the steps of SDS-Page
SDS coats proteins with a negative charge. An electric current is then applied, smaller proteins travel through the polyacrylamide gel toward the POSITIVE anode faster than larger ones, creating lanes of size separated protein bands
___ is a technique used to separate liquid molecules based on their boiling points, compounds with the LOWEST boiling point evaporate first
Distillation
When would you use simple distillation, fractional distillation, or vacuum distillation?
Simple distillation : bp<150 C and >25C
Fractional distillation: bp <25C
Vacuum Distillation: bp> 150C
___ separates molecules based on boiling point.
Intermolecular forces, molecular weight, and branching all affect boiling point. For compounds with same functional group but different carbon counts, higher molecular weight equates to a higher boiling point and longer retention time because of strong interaction with liquid phase
Gas-liquid chromatography
___ is a technique that separates components of a mixture based on polarity, polar molecules gave a higher affinity for the plate and travel up smaller distance than nonpolar molecules
Thin Layer Chromatography
___ is a type of chromatography in which a liquid solvent (mobile phase) carries the sample through a column filled with adsorbent material (stationary phase)
Molecules with polarity similar to the stationary phase have longer retention time than molecules with polarities similar to the mobile phase
High Performance liquid chromatography (HPLC)
A biochemical technique that utilizes antigen-antibody interactions to determine the presence of either
- antigens (proteins or cytokines)
- Specific immunoglobulins (antibodies)
Enzyme-linked immuno-sorbent assay (ELISA)
give the 6 steps of ELISA?
- Experimental wells are coated with antibodies for target antigen
- Sample of serum or cell extract is added to wells
- Antibodies immobilize the antigen by binding to it
- Any unbound proteins remaining in sample are washed away
- Enzyme-linked antibody that also recognizes target proteins are added to wells
- The wells are filled with a solution that changes color in presence of the detection enzyme.
A color change indicates target protein was present in sample
No color change means protein was absent
___ are used more extensively in the medical field to measure the relative amounts of hormones or drugs in patients’ serum
Radioimmunoassay (RIA)
5 steps of Radioimmunoassay
Step 1. A known amount of radiolabeled antigen (example: insulin that was synthesized with Iodine 25-labeled tyrosines) is incubated with a known amount of antibody that is specific to that antigen
Step 2. The insulin:antibody complexes are isolated
Step 3. The total amount of radioactivity is measured
Step 4. Unlabeled insulin is mixed into the solution in increasing amounts. The unlabeled insulin competes with the labeled insulin for the antibody. As more unlabeled insulin is added, less total radioactivity is recovered and measured. This competition assay helps formulate a standard curve.
Step 5. Steps 1-3 are repeated using patient serum instead of the unlabeled insulin. The standard curve is used to extrapolate the amount of insulin that is circulating in a patient’s serum
___ is a means of separating things (nucleic acids, proteins, etc.) by size or by charge.
Electrophoresis
How do things in Gel Electrophoresis move?
Things move through the gel toward positive pole, according to size; smaller things migrate faster (they fit more easily through the pores of the gel) and larger things migrate more slower)
