STEPTRACKERBIOCHEMISTRY + IMMUNOLOGY + GENETICS | LABORATORY TECHNIQUES | HIGH-YIELD REVIEW

Protein, Cell & Cytogenetic Techniques

Detect proteins with antibodies, sort cells by their surface markers, and look at chromosomes from whole karyotypes down to single genes.

FOCUS ON
ELISA detects antigen or antibody with an enzyme-linked antibody (screening); Western blot separates proteins by size (confirmation). Flow cytometry counts cells by fluorescent surface markers (CD4 count, leukemia typing, PNH). Karyotype = whole chromosomes in metaphase; FISH = probe for a specific locus (microdeletions, translocations). Knock-out / knock-in / transgenic mice test gene function.
TECHNIQUE
HOW IT WORKS
USE
CLUE
ELISA
Antigen or antibody captured on a plate; enzyme-linked antibody makes a color change
HIV screening, hepatitis serology, hormone levels
Very sensitive → screening; false positives possible
Western blot
Proteins separated by size (SDS-PAGE) → membrane → labeled antibody
Confirm a protein; Lyme disease confirmation
Western = protein
Flow cytometry
Single cells stream past lasers; fluorescent antibodies to surface/intracellular markers
CD4 count, lymphoma/leukemia immunophenotype, PNH (↓ CD55/CD59), fetal-maternal hemorrhage
Two-axis plots: each dot = one cell
Immunohistochemistry
Antibody stains a protein in a tissue section
Tumor origin (cytokeratin, S-100, desmin…), HER2, ER/PR
Keeps tissue architecture
Immunofluorescence
Fluorescent antibodies — direct (patient tissue) or indirect (patient serum)
Pemphigus, pemphigoid, glomerulonephritis patterns, ANA
Linear vs granular patterns
Karyotyping
Cells arrested in metaphase (colchicine), stained (G-banding), chromosomes arranged by size
Aneuploidy (trisomies), large deletions, translocations
Needs dividing cells; misses small deletions
FISH
Fluorescent DNA probe binds its matching locus on interphase or metaphase chromosomes
Microdeletions (22q11, Williams), translocations (BCR-ABL), HER2 amplification
No spot = deletion; extra spot = duplication
Chromosomal microarray
Compares patient vs reference DNA across the genome
Copy-number variants, first-line in developmental delay
Can't see balanced translocations
Knock-out mouse
Target gene removed or disabled
Learn what happens without the gene
Conditional knock-out = only in chosen tissue/time (Cre-lox)
Knock-in / transgenic mouse
Gene inserted at a set locus (knock-in) or randomly (transgenic)
Model human mutations; overexpression
—
Mass spectrometry & electrophoresis
Serum/urine protein electrophoresis, immunofixation; MS identifies proteins and metabolites
M spike in myeloma; newborn metabolic screen (tandem MS)
Newborn screen for PKU, MCAD, galactosemia…

Mechanism — sandwich ELISA

Capture antibodyOn plate
→
Patient antigenBinds
→
Enzyme-linked antibody
→
Substrate → colorIntensity ∝ amount

Indirect ELISA flips it: antigen on the plate catches the patient's antibody (e.g., HIV antibody test).

Karyotype vs FISH vs microarray

METHOD
SEES
MISSES
BEST FOR
Karyotype
Whole chromosomes, balanced translocations
Small deletions
Trisomy, Turner
FISH
One targeted locus
Everything not probed
22q11, BCR-ABL
Microarray
Copy-number genome-wide
Balanced rearrangements
Unexplained delay

How it's tested

COMMON CONFUSIONELISA screens (high sensitivity); Western blot confirms (high specificity). For HIV, current algorithms confirm with an antibody-differentiation assay and PCR.
THE TRAPA normal karyotype doesn't exclude DiGeorge — the 22q11 deletion is too small; use FISH or microarray.
YOUR APPROACHProtein, cell or chromosome? Screening or confirmation? Whole genome or one locus? → method.

If you see… think…

Measuring CD4 count → flow cytometry
RBCs lacking CD55/CD59 → flow cytometry for PNH
Screening test with an enzyme-linked antibody → ELISA
Proteins separated by size then probed → Western blot
Absent signal on one chromosome 22 with a probe → FISH for 22q11 deletion
Cells arrested in metaphase, G-banded → karyotype
Gene deleted only in liver of a mouse → conditional knock-out (Cre-lox)
M spike on serum electrophoresis → monoclonal gammopathy
Newborn heel-prick screen for amino acid disorders → tandem mass spectrometry
Tumor stained for cytokeratin in tissue → immunohistochemistry