Electrophoresis in the Lab

What does electrophoresis separate?

Charged molecules in an electric field

Where do cations migrate towards?

Cathodes

Where do anions migrate towards?

Anode

What is migration affected by?

Net charge

Molecular size/shape

Strength of the electric field

Properties of the support medium

Buffer pH/ionic strength

Temp

Greater charge =

Generally greater migration

Large molecule =

Generally slower migration

Proteins are ampholytes

They can carry positive or negative depending on pH

If pI is greater than pH then

Protein becomes more positively charged and will migrate toward the cathode

If pH is greater than pI then

Protein becomes more negatively charged, migrates toward the anode

If pH = pI, then

Net charge = 0

Electrophoretic migration stops

What does the buffer do?

Carries current and maintains pH, pH determines the charge on the proteins and therefore affects migration

What is the purpose of the support/separation medium?

Provides the environment in which molecules separate.

Electrodes and power supply do what in electrophoresis?

Create the electric field that drives migration

Detection and quantification system

Separated proteins can be stained or detected optically

A densitometer converts separated proteins zones into peaks that can be quantified

Agarose is what?

Predominantly charge-based separation

Polyacrylamide is what?

Based on charge and molecular size

Agarose - what should MLS students know?

Commonly used for serum protein electrophoresis; large pores, so proteins separate primarily according to charge

Polyacrylamide - what should MLS students know?

Smaller/tunable pores; separates based on charge and size b/c of molecular sieving

Cellulose acetate - what should MLS students know?

Older/historical support medium; largely replaced by agarose for many applications

Capillary - what should MLS students know?

Separation occurs inside a narrow capillary; rapid, automated, high-resolution method

Explain the process of detection and densitometry:

After conventional gel electrophoresis, separated proteins form bands or zones

Bands may be stained so they can be visualized

A densitometer measures absorbance across the separated zones

The resulting graph is an electropherogram

Area under each peak is proportional to the relative amount of protein in that fraction

Monoclonal gammopathy pattern

Narrow spike in the gamma region

Polyclonal inflammation pattern

Broad gamma-region increase

Nephrotic syndrome pattern

low albumin w/ increased a2 region

Cirrhosis pattern

Beta-gamma bridging

Hypogammaglobulinemia

Decreased gamma region

Where does capillary electrophoresis (CE) perform electrophoresis separation?

A very narrow capillary

Why does capillary electrophoresis use a high voltage?

The small capillaries dissipate heat efficiently

What are the advantages of capillary separation?

Rapid separation, high resolution, very small sample volume, automation, online detection

Electroosmotic flow

Capillary zone electrophoresis, buffer movement through the capillary helps carry analytes toward the detector

Analytes still separate b/c they have different electrophoresis mobilities

IFE separates proteins according to what?

Their isoelectric points

A pH gradient is established across what?

A pH gradient is established across the MEDIUM

Each protein migrates until what?

pH = protein's pI

When the pH = pI the net charge becomes what? And what happens to migration?

Net charge becomes 0

Migration stops

What will happen to proteins once they stop moving and net charge is zero?

Proteins become focused into sharp bands

What are Southern blots used to detect?

DNA

What are Northern blots used to detect?

RNA

What are Western blots used to detect?

Protein

How are separated molecules transferred in blotting techniques?

Transferred from a gel to a membrane

Which blotting techniques are uncommon? Why are they uncommon?

Southern and Northern blotting, they have been replaced by PCR, sequencing, and other molecular techniques

Western blotting has been replaced by what?

Newer immunoassays and molecular tests

Which blotting technique is historically important?

Western

Too much sample?

Overloaded bands or poor resolution

Too little sample

Faint bands or poor sensitivity

Wrong sample type

Plasma fibrinogen can add an extra band

Heat/current problems

Band broadening or distorted migration

Buffer problem?

pH/ionic strength changes migration

Uneven wetting/electrodes?

Unequal migration across gel

Stain/detection problem?

Weak or unreliable quantification

Point-of-application artifact

Band at the origin can mimic abnormally

What should you ask before interpreting a strange band?

Is it artifact or true abnormal protein?