Proteins are separated, transferred, the membrane is blocked, incubated with antibodies, then detected.
Incorrect! Try again.
19Western blotting is primarily used to determine the presence and approximate size of a:
Detection of proteins by western blotting
Easy
A.Specific protein
B.Lipid molecule
C.Specific gene
D.mRNA transcript
Correct Answer: Specific protein
Explanation:
Western blotting confirms whether a specific protein is present and estimates its molecular weight.
Incorrect! Try again.
20Why is a secondary antibody often conjugated to an enzyme in western blotting?
Detection of proteins by western blotting
Easy
A.To separate proteins
B.To block the membrane
C.To transfer proteins
D.To generate a detectable signal
Correct Answer: To generate a detectable signal
Explanation:
The enzyme on the secondary antibody catalyzes a reaction that produces a visible or luminescent signal.
Incorrect! Try again.
21In a western blot workflow, proteins separated by SDS-PAGE are transferred to a membrane. Which property of the proteins primarily determines their migration during the preceding electrophoresis step?
Detection of proteins by western blotting
Medium
A.Isoelectric point
B.Hydrophobicity
C.Molecular weight
D.Native charge
Correct Answer: Molecular weight
Explanation:
SDS coats proteins with a uniform negative charge, so migration in SDS-PAGE depends almost entirely on molecular weight rather than intrinsic charge or shape.
Incorrect! Try again.
22A researcher observes that proteins are not transferring efficiently from the gel to the membrane during electrotransfer. Which adjustment is most likely to improve transfer of a high molecular weight protein ( kDa)?
Detection of proteins by western blotting
Medium
A.Increasing methanol concentration sharply
B.Using a thicker membrane pore size of
C.Increasing transfer time and adding SDS to the buffer
D.Reducing the current to near zero
Correct Answer: Increasing transfer time and adding SDS to the buffer
Explanation:
Large proteins transfer slowly; extending transfer time and adding a small amount of SDS improves their elution from the gel. High methanol actually hinders large-protein transfer.
Incorrect! Try again.
23Why is a blocking step (e.g., with BSA or non-fat milk) performed after protein transfer in western blotting?
Detection of proteins by western blotting
Medium
A.To occupy unbound sites on the membrane and reduce non-specific antibody binding
B.To strip residual SDS from the proteins
C.To denature the transferred proteins completely
D.To crosslink proteins covalently to the membrane
Correct Answer: To occupy unbound sites on the membrane and reduce non-specific antibody binding
Explanation:
Blocking saturates the membrane's remaining protein-binding sites so antibodies bind only their specific target, lowering background signal.
Incorrect! Try again.
24In an indirect detection western blot, the primary antibody binds the target protein and the secondary antibody carries the enzyme label. What is the main advantage of this indirect approach over direct detection?
Detection of proteins by western blotting
Medium
A.It avoids any cross-reactivity entirely
B.It eliminates the need for a blocking step
C.Signal amplification because multiple secondary antibodies bind one primary
D.It removes the need for gel electrophoresis
Correct Answer: Signal amplification because multiple secondary antibodies bind one primary
Explanation:
Several labeled secondary antibodies can bind a single primary antibody, amplifying the detectable signal for greater sensitivity.
Incorrect! Try again.
25A western blot uses an HRP-conjugated secondary antibody. Which substrate/detection method is appropriate for this enzyme?
Detection of proteins by western blotting
Medium
A.Fluorescence using a UV-excited fluorophore
B.Radiolabeling with
C.Colorimetric reaction with -galactosidase substrate
D.Chemiluminescence using luminol substrate
Correct Answer: Chemiluminescence using luminol substrate
Explanation:
Horseradish peroxidase (HRP) oxidizes luminol to produce light, making chemiluminescence the standard readout for HRP-labeled antibodies.
Incorrect! Try again.
26A blot shows a strong band at the expected size but also multiple additional bands at other molecular weights. What is the most likely cause?
Detection of proteins by western blotting
Medium
A.Complete blocking of the membrane
B.Non-specific antibody binding or protein degradation
C.Overly stringent washing conditions
D.Insufficient primary antibody concentration
Correct Answer: Non-specific antibody binding or protein degradation
Explanation:
Extra bands typically arise from antibody cross-reactivity with other proteins or from degradation/proteolysis of the target, producing multiple fragments.
Incorrect! Try again.
27Which membrane property makes PVDF a common choice over nitrocellulose in western blotting?
Detection of proteins by western blotting
Medium
A.Ability to conduct electric current
B.Higher mechanical strength and protein binding capacity
C.Selective binding of only glycoproteins
D.Complete transparency to visible light
Correct Answer: Higher mechanical strength and protein binding capacity
Explanation:
PVDF membranes are more durable and have higher protein-binding capacity, allowing reprobing and better retention, though they require methanol activation.
Incorrect! Try again.
28During western blotting, a loading control such as -actin or GAPDH is probed. What is its primary purpose?
Detection of proteins by western blotting
Medium
A.To normalize protein loading across lanes
B.To confirm the transfer buffer pH
C.To verify membrane pore size
D.To measure antibody affinity directly
Correct Answer: To normalize protein loading across lanes
Explanation:
Housekeeping proteins are expressed at relatively constant levels, so they serve as a reference to confirm equal loading and to normalize target protein quantification.
Incorrect! Try again.
29A researcher gets a completely blank blot with no bands, including the loading control. Which explanation is most consistent with this result?
Detection of proteins by western blotting
Medium
A.Excessive target protein loaded
B.Failed protein transfer or missing/incorrect primary antibody
C.Over-exposure of the film
D.Too many wash steps only around the target band
Correct Answer: Failed protein transfer or missing/incorrect primary antibody
Explanation:
A total absence of signal, including the control, points to a systemic failure such as no transfer, omitted antibody, or an inactive detection reagent, rather than a target-specific problem.
Incorrect! Try again.
30Why are wash steps with buffers containing detergent (e.g., Tween-20 in TBST) performed between antibody incubations?
Detection of proteins by western blotting
Medium
A.To denature the target protein further
B.To permanently fix antibodies to the target
C.To remove unbound and weakly bound antibodies, reducing background
D.To increase membrane protein-binding capacity
Correct Answer: To remove unbound and weakly bound antibodies, reducing background
Explanation:
Detergent-containing washes strip away excess and non-specifically bound antibody, sharpening specific signal and lowering background noise.
Incorrect! Try again.
31A protein has a predicted molecular weight of 50 kDa but consistently appears at 65 kDa on a western blot. Which explanation is most plausible?
Detection of proteins by western blotting
Medium
A.The transfer buffer contained no methanol
B.The antibody is not specific to the protein
C.Post-translational modification such as glycosylation
D.The gel percentage was too high
Correct Answer: Post-translational modification such as glycosylation
Explanation:
Modifications like glycosylation or phosphorylation add mass and can alter migration, causing a protein to run higher than its predicted molecular weight.
Incorrect! Try again.
32Which sequence correctly represents the standard western blotting workflow?
Proteins are first separated by SDS-PAGE, transferred to a membrane, blocked, probed with primary then secondary antibody, and finally detected.
Incorrect! Try again.
33A researcher wants to detect a phosphorylated form of a protein specifically. Which strategy is most appropriate?
Detection of proteins by western blotting
Medium
A.Increase blocking with phosphoprotein-rich milk
B.Boil the sample longer to expose phosphate groups
C.Use a lower gel percentage to separate phosphates
D.Use a phospho-specific primary antibody
Correct Answer: Use a phospho-specific primary antibody
Explanation:
Phospho-specific antibodies recognize the phosphorylated epitope only. Note that milk contains casein (a phosphoprotein), so BSA is preferred for blocking in phospho-blots.
Incorrect! Try again.
34The purpose of including a pre-stained molecular weight ladder in a western blot is primarily to:
Detection of proteins by western blotting
Medium
A.Estimate the size of detected protein bands and monitor transfer
B.Provide the enzyme substrate for detection
C.Serve as the positive control for the antibody
D.Block non-specific binding sites
Correct Answer: Estimate the size of detected protein bands and monitor transfer
Explanation:
A pre-stained ladder migrates with known molecular weights, letting you estimate band sizes and visually confirm successful transfer to the membrane.
Incorrect! Try again.
35High background across the entire blot with a smeary appearance is observed. Which corrective action is most appropriate?
Detection of proteins by western blotting
Medium
A.Increase blocking time and dilute the antibodies further
B.Increase primary antibody concentration
C.Reduce the number of wash steps
D.Shorten the blocking step significantly
Correct Answer: Increase blocking time and dilute the antibodies further
Explanation:
High diffuse background usually reflects insufficient blocking or too-concentrated antibody; improving blocking and increasing antibody dilution reduces non-specific signal.
Incorrect! Try again.
36In quantitative western blotting, fluorescent secondary antibodies are often preferred over enzyme-based chemiluminescence because they:
Detection of proteins by western blotting
Medium
A.Offer a wider linear dynamic range for quantification
B.Cannot be affected by exposure time
C.Require no primary antibody
D.Eliminate the need for gel electrophoresis
Correct Answer: Offer a wider linear dynamic range for quantification
Explanation:
Fluorescent detection has a broader linear range and is more stable than enzymatic chemiluminescence, making band intensity more reliably proportional to protein amount.
Incorrect! Try again.
37Why must PVDF membranes be pre-wetted in methanol before use, unlike nitrocellulose?
Detection of proteins by western blotting
Medium
A.To increase its transparency for imaging
B.To reduce its protein-binding capacity
C.To activate the hydrophobic membrane so it can bind protein
D.To create pores in the membrane
Correct Answer: To activate the hydrophobic membrane so it can bind protein
Explanation:
PVDF is highly hydrophobic; methanol wets and activates the surface, enabling proper protein binding during transfer. Nitrocellulose does not require this step.
Incorrect! Try again.
38A weak or faint signal is obtained for a genuinely low-abundance target protein. Which change would best enhance detection sensitivity?
Detection of proteins by western blotting
Medium
A.Reduce protein loading in each lane
B.Increase detergent concentration in wash buffer
C.Skip the primary antibody incubation
D.Use a high-sensitivity ECL substrate and increase exposure time
Correct Answer: Use a high-sensitivity ECL substrate and increase exposure time
Explanation:
For low-abundance targets, switching to an enhanced chemiluminescent substrate and increasing exposure boosts sensitivity without changing sample amount.
Incorrect! Try again.
39What is the main reason samples are boiled with a reducing agent (e.g., -mercaptoethanol) and SDS before loading in a standard western blot?
Detection of proteins by western blotting
Medium
A.To conjugate the antibody to the protein
B.To label proteins with a fluorophore
C.To crosslink proteins to the membrane
D.To denature proteins and break disulfide bonds for size-based separation
Correct Answer: To denature proteins and break disulfide bonds for size-based separation
Explanation:
Reducing agents cleave disulfide bonds and SDS with heat denatures proteins into linear polypeptides, ensuring separation by size rather than conformation.
Incorrect! Try again.
40A researcher wants to reprobe the same membrane for a second protein of similar molecular weight to the first. What must be done before applying the new antibody?
Detection of proteins by western blotting
Medium
A.Strip the membrane to remove existing antibodies
B.Add fresh blocking buffer only, without stripping
C.Re-run SDS-PAGE on the membrane
D.Increase the transfer voltage
Correct Answer: Strip the membrane to remove existing antibodies
Explanation:
Stripping removes previously bound antibodies so the membrane can be reprobed. Without stripping, residual signal from the first probe would confound results at overlapping sizes.
Incorrect! Try again.
41During semi-dry transfer of a high molecular weight protein ( kDa) from an SDS-PAGE gel to a PVDF membrane, the transfer efficiency is poor. Which combination of adjustments is MOST likely to improve transfer of this large protein?
Detection of proteins by western blotting
Hard
A.Using nitrocellulose instead of PVDF and increasing methanol to 30%
B.Increasing methanol concentration to 20% and extending transfer time
C.Reducing SDS to trace amounts, lowering methanol concentration, and extending transfer time
D.Adding more SDS to the transfer buffer and increasing voltage sharply
Correct Answer: Reducing SDS to trace amounts, lowering methanol concentration, and extending transfer time
Explanation:
Methanol promotes protein-membrane binding but shrinks the gel and hinders elution of large proteins. Low methanol plus trace SDS keeps large proteins mobile, and longer transfer time allows complete migration out of the gel.
Incorrect! Try again.
42A researcher observes strong, uniform background across the entire blot in a chemiluminescent detection despite adequate blocking. The primary antibody was used at and secondary HRP-conjugate at . What is the MOST probable cause?
Detection of proteins by western blotting
Hard
A.Insufficient transfer of protein to the membrane
B.The membrane was over-exposed to transfer buffer
C.The blocking buffer contained too much detergent
D.Antibody concentrations are too high, causing nonspecific binding
Correct Answer: Antibody concentrations are too high, causing nonspecific binding
Explanation:
Uniform high background across the whole blot typically results from excess antibody binding nonspecifically. Diluting both primary and secondary antibodies usually reduces background.
Incorrect! Try again.
43In a western blot, a protein with a calculated molecular mass of kDa consistently migrates at an apparent mass of kDa. Assuming the protein is intact, which explanation is MOST consistent with this observation?
Detection of proteins by western blotting
Hard
A.Degradation of the protein during sample preparation
C.Post-translational modification such as glycosylation altering migration
D.Excessive SDS binding reducing apparent mass
Correct Answer: Post-translational modification such as glycosylation altering migration
Explanation:
Glycosylation and other modifications add mass and can alter SDS binding, causing anomalous migration at a higher apparent molecular weight than predicted from sequence.
Incorrect! Try again.
44Why is Ponceau S staining commonly performed immediately after transfer but before blocking in a western blot workflow?
Detection of proteins by western blotting
Hard
A.To permanently label the target protein for detection
B.To reversibly verify transfer efficiency and equal loading before antibody probing
C.To block nonspecific binding sites on the membrane
D.To cross-link proteins covalently to the membrane
Correct Answer: To reversibly verify transfer efficiency and equal loading before antibody probing
Explanation:
Ponceau S is a reversible total-protein stain that confirms successful transfer and loading without interfering with subsequent antibody-based detection, as it washes off during blocking.
Incorrect! Try again.
45A blot probed for a phosphoprotein shows no signal, though total protein controls are normal. The lab used milk as the blocking agent. What is the MOST likely reason for the missing phospho-signal?
Detection of proteins by western blotting
Hard
A.Milk prevents transfer of phosphorylated proteins
B.Milk denatures the phospho-epitope irreversibly
C.Milk contains phosphoproteins (casein) that mask phospho-specific antibody binding
D.Milk increases nonspecific HRP activity, quenching the signal
Casein in milk is phosphorylated and competes with phospho-specific antibodies, suppressing signal. BSA is the preferred blocking agent for phospho-detection.
Incorrect! Try again.
46In enhanced chemiluminescence (ECL), HRP catalyzes oxidation of luminol in the presence of . If a blot produces an extremely intense signal that rapidly fades within seconds, which corrective action is MOST appropriate?
Detection of proteins by western blotting
Hard
A.Increase exposure time on the imager
B.Increase the secondary antibody concentration further
C.Dilute the secondary antibody to reduce enzyme load and slow substrate consumption
D.Add more to the substrate solution
Correct Answer: Dilute the secondary antibody to reduce enzyme load and slow substrate consumption
Explanation:
Rapid burnout indicates too much HRP consuming substrate quickly. Reducing antibody concentration lowers enzyme density, producing a slower, more sustainable signal.
Incorrect! Try again.
47A western blot displays a distinct 'smiling' pattern where bands curve upward at the edges of the gel lanes, later reflected in the blot. This artifact originates from which condition during electrophoresis?
Detection of proteins by western blotting
Hard
A.Uneven heat distribution causing faster migration in the center
B.Air bubbles trapped during transfer
C.Incomplete polymerization of the stacking gel
D.Overloading of protein in the central lanes
Correct Answer: Uneven heat distribution causing faster migration in the center
Explanation:
The 'smiling' effect results from the center of the gel heating more than the edges during electrophoresis, accelerating central migration. Lower voltage or better cooling prevents it.
Incorrect! Try again.
48When comparing nitrocellulose and PVDF membranes for western blotting, which statement correctly describes a KEY functional difference relevant to reprobing?
Detection of proteins by western blotting
Hard
A.PVDF has higher protein-binding capacity and greater durability for reprobing
B.Nitrocellulose has higher mechanical strength and tolerates repeated stripping
C.PVDF requires no methanol activation and binds less protein
Correct Answer: PVDF has higher protein-binding capacity and greater durability for reprobing
Explanation:
PVDF has higher binding capacity ( µg/cm²) and superior mechanical strength, making it more suitable for stripping and reprobing than the more fragile nitrocellulose.
Incorrect! Try again.
49A researcher wants to detect two proteins of similar molecular weight ( and kDa) on the same blot using antibodies raised in the same host species (both rabbit). What is the MOST reliable strategy to distinguish them?
Detection of proteins by western blotting
Hard
A.Increase gel percentage to fully resolve them without stripping
B.Use two different HRP substrates at once
C.Strip the membrane between probings and detect sequentially
D.Probe simultaneously and rely on band separation alone
Correct Answer: Strip the membrane between probings and detect sequentially
Explanation:
Because both antibodies share the host species, a single secondary antibody would bind both. Stripping and sequential detection isolates each signal, avoiding cross-reactivity confusion.
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50In a quantitative western blot, a loading control (e.g., -actin) is used for normalization. When is normalizing to a single housekeeping protein LEAST valid?
Detection of proteins by western blotting
Hard
A.When the target protein is highly abundant
B.When using fluorescent secondary antibodies
C.When samples are loaded in equal volumes
D.When the housekeeping protein expression changes under the experimental condition
Correct Answer: When the housekeeping protein expression changes under the experimental condition
Explanation:
Normalization assumes the loading control is invariant. If the housekeeping protein itself changes with treatment, it fails as a reference, and total-protein normalization becomes preferable.
Incorrect! Try again.
51A blot exhibits multiple bands where only a single band is expected. The antibody is polyclonal and validated. Which explanation is MOST consistent with the extra bands representing genuine biology rather than artifact?
Detection of proteins by western blotting
Hard
A.Bubbles disrupting transfer at those positions
B.Alternative splice variants or proteolytic isoforms of the target
C.Cross-reactivity with the blocking agent
D.Overloaded lanes causing streaking
Correct Answer: Alternative splice variants or proteolytic isoforms of the target
Explanation:
Multiple specific bands can reflect real isoforms, splice variants, or processed forms of the target protein, especially with polyclonal antibodies recognizing multiple epitopes.
Incorrect! Try again.
52The Towbin transfer buffer classically contains Tris, glycine, and methanol. What is the primary electrochemical role of glycine in this buffer during electrotransfer?
Detection of proteins by western blotting
Hard
A.It denatures proteins to improve mobility
B.It covalently binds proteins to the membrane
C.It stabilizes HRP activity during transfer
D.It acts as a trailing ion in the moving boundary system
Correct Answer: It acts as a trailing ion in the moving boundary system
Explanation:
Glycine functions as the slow-migrating trailing ion in the discontinuous buffer system, creating the moving boundary that drives proteins toward the membrane.
Incorrect! Try again.
53A fluorescent western blot using near-infrared detection shows signal saturation for the target but a linear signal for the loading control. How does this affect quantification, and what is the correct remedy?
Detection of proteins by western blotting
Hard
A.Saturation inflates target signal; load less sample or reduce exposure to stay in the linear range
B.Saturation affects only the control; reload the control lane
C.Saturation deflates the target signal; increase antibody concentration
D.Saturation has no effect; proceed with the ratio as measured
Correct Answer: Saturation inflates target signal; load less sample or reduce exposure to stay in the linear range
Explanation:
Saturated pixels underreport true abundance because signal plateaus, distorting the target-to-control ratio. Quantification requires all signals to fall within the linear dynamic range.
Incorrect! Try again.
54During stripping and reprobing, harsh stripping buffers (low pH, high SDS, reducing agents) can compromise results. Which outcome is the MOST likely consequence of over-stripping a membrane?
Detection of proteins by western blotting
Hard
A.Loss of target protein from the membrane, reducing subsequent signal
B.Increased nonspecific background on reprobing
C.Complete blocking of all binding sites
D.Covalent cross-linking of remaining antibodies
Correct Answer: Loss of target protein from the membrane, reducing subsequent signal
Explanation:
Aggressive stripping not only removes antibodies but can elute the bound target protein itself, weakening or eliminating the signal on subsequent probing.
Incorrect! Try again.
55A membrane probed with a mouse monoclonal primary and an anti-mouse HRP secondary shows strong bands at and kDa in samples that are immunoprecipitates, unrelated to the target. What is the MOST likely source of these bands?
Detection of proteins by western blotting
Hard
A.Nonspecific binding of the primary antibody to the membrane
B.Degradation products of the target protein
C.Cross-reactivity with the blocking milk proteins
D.Detection of the heavy and light chains of IP antibodies by the secondary antibody
Correct Answer: Detection of the heavy and light chains of IP antibodies by the secondary antibody
Explanation:
IP antibodies denature into heavy ( kDa) and light ( kDa) chains, which the anti-mouse secondary detects. Using conformation-specific or clean-blot reagents avoids this.
Incorrect! Try again.
56In a two-color fluorescent western blot, spectral bleed-through between channels is suspected. Which control BEST confirms whether cross-channel signal is genuine or an artifact?
Detection of proteins by western blotting
Hard
A.Imaging single-antibody controls in each channel separately
B.Adding excess blocking agent to both channels
C.Running a blank membrane with no antibodies
D.Increasing exposure equally in both channels
Correct Answer: Imaging single-antibody controls in each channel separately
Explanation:
Single-label controls reveal whether a signal from one fluorophore appears in the other channel, distinguishing true co-detection from spectral bleed-through.
Incorrect! Try again.
57A researcher notices that a hydrophobic membrane protein transfers poorly and aggregates near the top of the gel. Which sample-preparation adjustment MOST directly addresses this?
Detection of proteins by western blotting
Hard
A.Avoiding boiling and instead heating gently () to prevent aggregation
B.Adding more reducing agent and boiling twice
C.Boiling the sample longer at to fully denature it
D.Increasing the SDS concentration and boiling at
Correct Answer: Avoiding boiling and instead heating gently () to prevent aggregation
Explanation:
Boiling hydrophobic membrane proteins promotes irreversible aggregation. Gentle heating retains solubility and improves entry into the gel and subsequent transfer.
Incorrect! Try again.
58Signal appears only at the edges of the membrane and is absent in the center after chemiluminescent detection. Assuming even protein loading and transfer, what is the MOST likely cause?
Detection of proteins by western blotting
Hard
A.Overexposure of the central region
B.Uneven substrate contact or drying of the central membrane region
C.Excess blocking in the center only
D.Antibody degradation during incubation
Correct Answer: Uneven substrate contact or drying of the central membrane region
Explanation:
If the membrane dries or the ECL substrate does not evenly coat the surface, central regions lose signal. Ensuring uniform, continuous substrate coverage prevents this.
Incorrect! Try again.
59For validating antibody specificity in western blotting, which control provides the STRONGEST evidence that a band represents the genuine target?
Detection of proteins by western blotting
Hard
A.A knockout or knockdown sample lacking the target protein showing band loss
B.A molecular weight marker matching the band
C.A no-primary-antibody control lane
D.A second gel run under identical conditions
Correct Answer: A knockout or knockdown sample lacking the target protein showing band loss
Explanation:
Disappearance of the band in a knockout/knockdown sample directly demonstrates that the antibody detects the intended target, the gold standard for specificity validation.
Incorrect! Try again.
60A researcher must detect a very low-abundance target and considers switching from HRP-chemiluminescence to a biotin-streptavidin amplified system. What is the primary trade-off of this amplification approach?
Detection of proteins by western blotting
Hard
A.Reduced sensitivity but faster protocol
B.No change in sensitivity but improved reproducibility
C.Increased sensitivity accompanied by higher risk of background from endogenous biotin
D.Lower background but reduced signal linearity
Correct Answer: Increased sensitivity accompanied by higher risk of background from endogenous biotin
Explanation:
Biotin-streptavidin systems amplify signal for low-abundance targets, but endogenous biotin in samples can bind streptavidin and elevate background, requiring additional blocking.
Incorrect! Try again.
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