Biochemistry Chapter 5: Analytic Techniques Flashcards
In gel electrophoresis, molecules move from
Cathode (-) to anode (+)
True or false: PCR can be used to quantify gene expression
False. Quantifying gene expression must be performed with other lab techniques, such as qPCR/RT-PCR.
what does PCR require?
Forward and reverse primers
Taq polymerase
A magnesium containing buffer
True or false: retention factor can only be between 0 - 1
True. Its Rf value will thus be quite high. Rf, or the retention factor, is equal to the distance a given solute has migrated up the stationary phase, divided by the maximum distance traveled by the mobile phase, or solvent. This means that Rf can range from 0 to 1; lower Rf values (closer to 0) are associated with more polar compounds, whereas higher values (closer to 1) are associated with more nonpolar compounds.
Rf formula
Beer lambert law
where A is absorptivity, ε is the molar attenuation coefficient, and c is the concentration. Essentially, it indicates that higher levels of absorbance mean that more light is absorbed, which means that more of a substance is present.
technique that assesses the concentration of protein by measuring the extent to which the unlabeled proteins in the sample compete with radioactively labeled antigens for antibody binding sites
Radioimmunoassay
technique in which antibodies bind antigens, and then the amount of bound antibody is determined by reactions that produce a visible signal, like color or fluorescence
ELISA
Rule of thumb for correlation between variables
Fractional distillation is used to separate what kind of compounds?
Compounds with very close boiling points
When to use thin layer chromatography vs gel electrophoresis
TLC = for organic (polar/non-polar)
Gel Electrophoresis = for charged species like DNA
what direction do the proteins migrate in SDS-PAGE?
Towards the positive anode (SDS-PAGE has a current running through it). Like an electrolytic cell
why is albumin often used in biochemical and enzymatic experiments?
In biochemical and enzymatic experiments, albumin (such as BSA) is commonly used to prevent enzyme adsorption to surfaces, stabilize proteins, and maintain reaction homogeneity for accurate kinetic measurements.
Native PAGE vs. SDS-PAGE
Native PAGE (without SDS) keeps proteins in their native, folded states based on charge and shape.
SDS-PAGE intentionally denatures proteins for accurate size measurement.
Protein folding is what level of structure?
tertiary structure
RNA folding vs Protein Folding
In native gel electrophoresis, ______________ plays the biggest role in RNA migration, because it determines how compact the molecule is.
Secondary structure (remember there is a difference between RNA folding and protein folding!)
Based on the data in Figure 3, what is the melting temperature Tm of wild-type M1 mRNA in a solution containing 100 mM KCl?
53C - Tm occurs when folded/unfolded is 0.5
What is found in the super natant vs the pellet?
Supernatant (SN): Contains cytoplasmic proteins. If cytochrome c is detected in the supernatant, it means it has been released from the mitochondria into the cytoplasm, indicating apoptosis.
Pellet: Contains intact organelles, including mitochondria. If cytochrome c is still detected in the pellet, it means it has not been released from the mitochondria, indicating that apoptosis has not been initiated.
in Isoelectric focusing (IEF), the cathode is (+ or o) and the anode is (+ or -)?
Cathode = (-)
Anode = (+)
Mock-transfected cells
✔ Mock-transfected cells are a negative control to rule out any effects from the transfection process itself.
✔ They undergo the same transfection procedure but do NOT receive the experimental gene.
When would you use immunohistochemistry and when would you use western blot?
