Lab FInal Review

Purpose of SDS in SDS-PAGE

Denatures proteins and gives uniform negative charge

Why do proteins separate by size in SDS-PAGE

Denatures proteins and gives uniform negative charge

Purpose of glycine in running buffer

Helps carry current during electrophoresis

Why should 5X running buffer not be pH adjusted

Buffer works at its natural pH

Purpose of glycerol in Laemmli buffer

Makes samples sink into wells

Purpose of bromophenol blue

Tracking dye during electrophoresis

Purpose of transfer buffer

Transfers proteins from gel to membrane

Purpose of Tween-20 in T-TBS

Reduces nonspecific binding

What does T-TBS stand for

Tween-Tris Buffered Saline

Formula for grams needed in solution prep

Grams = M*V*MW

Why convert mL to L in molarity problems

Molarity uses liters

Formula for dilutions

C1​V1​=C2​V2​

What does q.s. mean

Bring to final volume

Why calibrate a pH meter

Ensures accurate readings

Why rinse probe between solutions

Prevents contamination

Common calibration buffers

pH 4,7, and 10

Why store electrode in storage solution

Prevents probe damage/drying

What does a spectrophotometer measure

Light absorbance

Why blank the spectrophotometer

Sets baseline to zero

Why select proper wavelength

Molecules absorb best at specific wavelengths

Accuracy vs precision

Accuracy = correct values

Precision = reproducibility

Purpose of filter tips

Prevent contamination

First stop on pipette

Aspirates liquid

Second stop on pipette

Expels remaining liquid

What happens if plunger is released too fast

Bubbles/inaccurate volume

Air displacement pipette uses what mechanism

Partial vacuum

Positive displacement pipettes are best for what

Viscous or volatile liquids

What sterilizes in an autoclave

High-pressure steam

Why use slow exhaust for liquids

Prevents boiling over

Why leave bags loosely closed

Allows steam penetration

Why crack autoclave door first

Releases steam safely

Why wait for pressure to reach zero

Prevent burns/explosion risk

What does PBS stand for

Phosphate-buffered saline

What is the purpose of PBS

Maintains pH and salt balance

Why is 70% ethanol effective

Water improves protein denaturation

Most common pipette used in labs

Manual micropipette

What does the volume lock do on a micropipette

Prevents accidental volume changes

Purpose of tip ejector button

Removes disposable tip

Purpose of disposable pipette tip

Prevents contamination

Electronic pipettes use what instead of thumb plunger control

Trigger/button system

Electronic pipettes are useful for what

Repeat dispensing, titrations, serial dilutions

Multichannel pipettes are ideal for what

96-well ELISA and PCR plates

Multichannel pipettes can be what 2 types

Manual or electronic

Purpose of repeat pipettes

Dispense aliquots repeatedly from one apsiration

Automatic pipette controllers are used for what

Large liquid volumes

Automatic pipette controllers work with what type of pipettes

Glass or plastic serological pipettes

Eppendorf pipettes are used for what

Measuring small liquid volumes accurately

First stop on Eppendorf pipettes

Expels liquid completly

P10 range

0.1–10 µL

P20 range

2–20 µL.

P100 range

10–100 µL

P200 range

20–200 µL

P1000 range

200–1000 µL

Serological pipettes are mainly used in what environment

Steril environments like cell culture

Common serological pipette sizes

1,5,10,25 mL

Purpose of cotton stopper in serological pipette

Prevents contamination of pipette aide

What are pipette aides

Mechanical devices assisting liquid pipetting

Top button on pipette aide

Draws liquid up

Second button on pipette aide

Expels liquid

What happens if liquid reaches cotton stopper

Filter becomes wet and won’t draw liquid properly

What does standard deviation measure

Spread/variation in data

Low standard deviation means what

Data points are close together

Purpose of protein determination /bradford assay lab

Determine protein concentration in samples

What assay is used in the protein determination /bradford assay lab

Bio-Rad Protein

Bio-Rad assay is based on what method

Bradford method

Bradford assay measures absorbance at what wavelength

595 nm

Bradford assay uses what instrument

Spectrophotometer

What dye is used in Bradford assay

Coomassie Brilliant Blue G-250

Dye shifts from what wavelength to what wavelength after protein binding

465 nm → 595 nm

Coomassie dye binds mainly to what amino acid residues

Basic and aromatic resides

What is BSA

Bovine Serum Albumin

BSA comes from what source

Cow protein

BSA is used for what in Bradford assay

Protein standard for standard curve

Purpose of standard curve

Determine unknown protein concentration

Equation of a line for standard curve

y=mx+b

In ymx+b, what does x represent

Protein concentration

Formula to solve for unknown concentration

x = y-b / m

What does R2 represent

How well data fits the line

Ideal R2 value

1.0

Points far from line do what to R2

Lower it

Bio-Rad dye concentrate is diluted how

1 part dye : 4 parts ddH2O

Why make extra Bio-Rad reagent

Incase assay must be repeated / spilled

Why keep samples on ice

Preserve samples/proteins

Microsomal fractions were dilutes to what ration

1:30

Standard curve tubes should start with what addition order

Water → BSA → Bio-Rad dye

Standard curve blank contains how much BSA

0 µL

Highest standard concentration in curve

1.0 mg/mL

How much sample was added to each analysis tube from the Bradford protein assay experiment

100 µL

How much Bio-Rad dye was added to each tube

5.0 mL

Tubes 13 and 14 during this experiment were what

Buffer blanks

Samples were incubates for how long in the Bradford experiment

5 minutes

Bradford assay absorbance was measured at what wavelength

595 nm

After measuring absorbance, what is plotted

Standard curve

Unknown protein concentrations are calculated using what

Standard curve

What must be subtracted from unknown samples

Buffer blank values

A higher R2 means what

Better line fit

Bio-Rad assay can also be used with what instrument

Microplate reader

Dye reagent dilution microtiter assay

1 part dye : 4 parts ddH2O

What filter paper was used during protein assay

Whatman #1 filter

Microtiter assay samples were assayed in what

Quadruplicate