Western Blot Wisdom
What material is used to give proteins a negative charge for size-based separation
SDS
Which ELISA format is commonly used to measure protein concentration in samples?
Sandwich
What must you do to live cells before standard immunofluorescence staining?
Fixation and permeabilization
What scatter readout tracks cell size for gating?
FSC
What step moves proteins from gel to membrane before detection?
Transfer
What is one common sample type used for ELISA (e.g., serum, plasma, cell culture supernatant)?
Serum
In what situations should you use IF/IHC to measure protein expression? (name two)
When you need spatial localization (e.g., nucleus vs membrane) and cell/tissue context (e.g., tumor vs stroma or specific cell types)
What scatter readout tracks cell complexity or granularity?
SSC
What membrane type is commonly used for Western blotting due to good protein binding and easy handling?
Nitrocellulose
Which enzyme label is commonly used in ELISA to generate the color signal?
Horse-radish peroxidase
Which imaging technique best visualizes immunofluorescence staining?
Confocal microscopy
What sample-prep step is required to stain intracellular proteins (e.g., transcription factors, cytokines) to measure their expression?
Permeabilization
Multiple choice — When is Western blot best used to measure protein expression? (Choose all that apply)
A) To confirm the presence and approximate size of a specific protein in cell/tissue lysates.
B) To detect cleavage products or isoforms via band size shifts.
C) To compare relative changes of a small set of targets between conditions.
D) To verify knockdown/knockout or overexpression at the protein level.
E) To quantify thousands of proteins at once in an unbiased screen.
F) To get absolute pg/mL concentrations in serum/plasma with high sensitivity.
G) To measure protein expression at single-cell resolution across mixed populations.
H) To map spatial localization of a protein within tissue sections.
A, B, C, D.
When is ELISA best used to measure protein expression? (Choose all that apply)
A) Screening thousands of proteins at once in an unbiased way.
B) Getting absolute concentration for one analyte using a standard curve.
C) Measuring protein localization within tissues.
D) Comparing relative changes of a small panel (1–10) of secreted proteins.
E) Single-cell expression across mixed populations.
F) Rapid validation of a biomarker in many samples with moderate throughput.
G) Directly detecting intracellular proteins without lysis.
B, D, F
Is the statement correct?
IF/IHC can measure protein expression in situ and allow semi-quantitative comparisons if imaging settings and analysis are standardized
True
What background signal from cells or tissues can mask weak protein expression and reduce detection sensitivity?
Autofluorescence
Is the following statement correct? Choose one: True or False.
Statement: “Western blot is high-throughput and provides absolute concentrations across hundreds of proteins per run.”
False
Is the statement correct?
Statement: “ELISA is unbiased, high-plex (thousands of proteins), and does not require calibration.
False
Match each marker to its protein target.
Markers: AE1/AE3, DM1A, AC-15, V9.
Targets (choose from): pan-cytokeratin (CK), α-tubulin, β-actin (ACTB), vimentin (VIM)
AE1/AE3 → pan-cytokeratin (CK).
DM1A → α-tubulin.
AC-15 → β-actin (ACTB).
V9 → vimentin (VIM).
When is flow cytometry best used to measure protein expression? (Choose all that apply)
A) When you need single-cell protein expression across mixed populations.
B) When you need absolute pg/mL concentrations in serum/plasma.
C) When you need spatial localization in intact tissue sections.
D) When you need co-expression of multiple markers per cell (multiparameter panels).
E) When you want to profile thousands of proteins without antibodies.
F) When you need to sort live cells based on protein expression for downstream assays.
G) When your only material is FFPE (formalin-fixed, paraffin-embedded) archival tissue.
H) When measuring rapid, inducible intracellular proteins after short stimulations using fix/perm
A, D, F, H