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Unit 6: Detection of proteins by western blotting - Practice Quiz

BTY555 — Biotechnology Laboratory-I 60 Questions
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1 Western blotting is a technique used to detect specific:

Detection of proteins by western blotting Easy
A. DNA fragments
B. RNA molecules
C. Carbohydrates
D. Proteins

2 Which technique is used to detect DNA and is analogous to western blotting?

Detection of proteins by western blotting Easy
A. Dot blotting
B. Southern blotting
C. Eastern blotting
D. Northern blotting

3 Proteins are usually separated before western blotting using:

Detection of proteins by western blotting Easy
A. Agarose gel electrophoresis
B. SDS-PAGE
C. Column chromatography
D. Centrifugation

4 In western blotting, separated proteins are transferred from the gel onto a:

Detection of proteins by western blotting Easy
A. Glass slide
B. Petri dish
C. Filter paper only
D. Membrane

5 Which of the following membranes is commonly used in western blotting?

Detection of proteins by western blotting Easy
A. Cellulose acetate
B. PVDF
C. Alumina
D. Silica gel

6 The process of transferring proteins from a gel to a membrane is called:

Detection of proteins by western blotting Easy
A. Staining
B. Blotting
C. Probing
D. Blocking

7 In western blotting, target proteins are detected using:

Detection of proteins by western blotting Easy
A. Restriction enzymes
B. Antibodies
C. DNA probes
D. Enzymes only

8 The antibody that directly binds to the target protein is called the:

Detection of proteins by western blotting Easy
A. Primary antibody
B. Tertiary antibody
C. Secondary antibody
D. Blocking antibody

9 The secondary antibody in western blotting binds to the:

Detection of proteins by western blotting Easy
A. Primary antibody
B. Target protein directly
C. Membrane
D. Gel

10 The step used to prevent non-specific binding of antibodies is called:

Detection of proteins by western blotting Easy
A. Elution
B. Transfer
C. Blocking
D. Blotting

11 A commonly used blocking agent in western blotting is:

Detection of proteins by western blotting Easy
A. Ethanol
B. Non-fat dry milk
C. Glycerol
D. Sodium chloride

12 The enzyme commonly conjugated to secondary antibodies for detection is:

Detection of proteins by western blotting Easy
A. Amylase
B. Horseradish peroxidase
C. Lipase
D. DNA polymerase

13 Which detection method uses light emission to visualize proteins?

Detection of proteins by western blotting Easy
A. UV absorbance
B. Mass spectrometry
C. Autoradiography of DNA
D. Chemiluminescence

14 In SDS-PAGE used before western blotting, proteins are separated based on their:

Detection of proteins by western blotting Easy
A. Solubility
B. Molecular weight
C. Charge only
D. Color

15 A molecular weight marker (ladder) is used in western blotting to:

Detection of proteins by western blotting Easy
A. Transfer proteins
B. Block the membrane
C. Stain the gel
D. Estimate protein size

16 Which buffer additive commonly reduces background in wash steps of western blotting?

Detection of proteins by western blotting Easy
A. Glucose
B. Tween-20
C. Calcium chloride
D. Sucrose

17 Which method drives proteins from the gel to the membrane using electric current?

Detection of proteins by western blotting Easy
A. Electroblotting
B. Diffusion
C. Capillary action only
D. Centrifugation

18 The correct general order of western blotting steps is:

Detection of proteins by western blotting Easy
A. Detection → transfer → blocking → electrophoresis
B. Transfer → detection → blocking → electrophoresis
C. Electrophoresis → transfer → blocking → antibody incubation → detection
D. Blocking → detection → electrophoresis → transfer

19 Western blotting is primarily used to determine the presence and approximate size of a:

Detection of proteins by western blotting Easy
A. Specific gene
B. mRNA transcript
C. Specific protein
D. Lipid molecule

20 Why 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 generate a detectable signal
D. To transfer proteins

21 In 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. Native charge
B. Hydrophobicity
C. Molecular weight
D. Isoelectric point

22 A 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. Reducing the current to near zero
D. Increasing transfer time and adding SDS to the buffer

23 Why 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 denature the transferred proteins completely
C. To strip residual SDS from the proteins
D. To crosslink proteins covalently to the membrane

24 In 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 eliminates the need for a blocking step
B. It removes the need for gel electrophoresis
C. Signal amplification because multiple secondary antibodies bind one primary
D. It avoids any cross-reactivity entirely

25 A 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. Chemiluminescence using luminol substrate
B. Colorimetric reaction with -galactosidase substrate
C. Radiolabeling with
D. Fluorescence using a UV-excited fluorophore

26 A 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. Insufficient primary antibody concentration
B. Non-specific antibody binding or protein degradation
C. Complete blocking of the membrane
D. Overly stringent washing conditions

27 Which membrane property makes PVDF a common choice over nitrocellulose in western blotting?

Detection of proteins by western blotting Medium
A. Higher mechanical strength and protein binding capacity
B. Ability to conduct electric current
C. Selective binding of only glycoproteins
D. Complete transparency to visible light

28 During 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 confirm the transfer buffer pH
B. To normalize protein loading across lanes
C. To measure antibody affinity directly
D. To verify membrane pore size

29 A 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. Failed protein transfer or missing/incorrect primary antibody
B. Too many wash steps only around the target band
C. Excessive target protein loaded
D. Over-exposure of the film

30 Why 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 increase membrane protein-binding capacity
D. To remove unbound and weakly bound antibodies, reducing background

31 A 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 gel percentage was too high
B. The transfer buffer contained no methanol
C. Post-translational modification such as glycosylation
D. The antibody is not specific to the protein

32 Which sequence correctly represents the standard western blotting workflow?

Detection of proteins by western blotting Medium
A. Transfer → SDS-PAGE → detection → block → antibodies
B. SDS-PAGE → transfer → block → primary antibody → secondary antibody → detection
C. Block → SDS-PAGE → transfer → detection → antibodies
D. Primary antibody → SDS-PAGE → block → transfer → detection

33 A 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

34 The 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. Block non-specific binding sites
C. Serve as the positive control for the antibody
D. Provide the enzyme substrate for detection

35 High 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. Shorten the blocking step significantly
B. Increase blocking time and dilute the antibodies further
C. Reduce the number of wash steps
D. Increase primary antibody concentration

36 In quantitative western blotting, fluorescent secondary antibodies are often preferred over enzyme-based chemiluminescence because they:

Detection of proteins by western blotting Medium
A. Require no primary antibody
B. Cannot be affected by exposure time
C. Offer a wider linear dynamic range for quantification
D. Eliminate the need for gel electrophoresis

37 Why must PVDF membranes be pre-wetted in methanol before use, unlike nitrocellulose?

Detection of proteins by western blotting Medium
A. To reduce its protein-binding capacity
B. To increase its transparency for imaging
C. To create pores in the membrane
D. To activate the hydrophobic membrane so it can bind protein

38 A 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. Increase detergent concentration in wash buffer
B. Use a high-sensitivity ECL substrate and increase exposure time
C. Skip the primary antibody incubation
D. Reduce protein loading in each lane

39 What 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 crosslink proteins to the membrane
B. To denature proteins and break disulfide bonds for size-based separation
C. To conjugate the antibody to the protein
D. To label proteins with a fluorophore

40 A 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. Increase the transfer voltage
C. Re-run SDS-PAGE on the membrane
D. Add fresh blocking buffer only, without stripping

41 During 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. Increasing methanol concentration to 20% and extending transfer time
B. Using nitrocellulose instead of PVDF and increasing methanol to 30%
C. Adding more SDS to the transfer buffer and increasing voltage sharply
D. Reducing SDS to trace amounts, lowering methanol concentration, and extending transfer time

42 A 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. Antibody concentrations are too high, causing nonspecific binding
B. The blocking buffer contained too much detergent
C. Insufficient transfer of protein to the membrane
D. The membrane was over-exposed to transfer buffer

43 In 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. Post-translational modification such as glycosylation altering migration
B. Incomplete denaturation leaving residual secondary structure
C. Excessive SDS binding reducing apparent mass
D. Degradation of the protein during sample preparation

44 Why 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 cross-link proteins covalently to the membrane
B. To block nonspecific binding sites on the membrane
C. To permanently label the target protein for detection
D. To reversibly verify transfer efficiency and equal loading before antibody probing

45 A 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 denatures the phospho-epitope irreversibly
B. Milk prevents transfer of phosphorylated proteins
C. Milk contains phosphoproteins (casein) that mask phospho-specific antibody binding
D. Milk increases nonspecific HRP activity, quenching the signal

46 In 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. Add more to the substrate solution
B. Increase exposure time on the imager
C. Increase the secondary antibody concentration further
D. Dilute the secondary antibody to reduce enzyme load and slow substrate consumption

47 A 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. Overloading of protein in the central lanes
B. Uneven heat distribution causing faster migration in the center
C. Air bubbles trapped during transfer
D. Incomplete polymerization of the stacking gel

48 When 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 requires no methanol activation and binds less protein
B. PVDF has higher protein-binding capacity and greater durability for reprobing
C. Nitrocellulose binds proteins covalently, allowing infinite reuse
D. Nitrocellulose has higher mechanical strength and tolerates repeated stripping

49 A 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. Probe simultaneously and rely on band separation alone
B. Increase gel percentage to fully resolve them without stripping
C. Strip the membrane between probings and detect sequentially
D. Use two different HRP substrates at once

50 In 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 samples are loaded in equal volumes
B. When the target protein is highly abundant
C. When the housekeeping protein expression changes under the experimental condition
D. When using fluorescent secondary antibodies

51 A 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. Cross-reactivity with the blocking agent
C. Alternative splice variants or proteolytic isoforms of the target
D. Overloaded lanes causing streaking

52 The 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 covalently binds proteins to the membrane
B. It stabilizes HRP activity during transfer
C. It denatures proteins to improve mobility
D. It acts as a trailing ion in the moving boundary system

53 A 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 deflates the target signal; increase antibody concentration
B. Saturation affects only the control; reload the control lane
C. Saturation has no effect; proceed with the ratio as measured
D. Saturation inflates target signal; load less sample or reduce exposure to stay in the linear range

54 During 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. Complete blocking of all binding sites
C. Covalent cross-linking of remaining antibodies
D. Increased nonspecific background on reprobing

55 A 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. Detection of the heavy and light chains of IP antibodies by the secondary antibody
B. Degradation products of the target protein
C. Nonspecific binding of the primary antibody to the membrane
D. Cross-reactivity with the blocking milk proteins

56 In 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. Running a blank membrane with no antibodies
B. Increasing exposure equally in both channels
C. Adding excess blocking agent to both channels
D. Imaging single-antibody controls in each channel separately

57 A 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. Increasing the SDS concentration and boiling at
C. Boiling the sample longer at to fully denature it
D. Adding more reducing agent and boiling twice

58 Signal 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

59 For 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 no-primary-antibody control lane
B. A molecular weight marker matching the band
C. A knockout or knockdown sample lacking the target protein showing band loss
D. A second gel run under identical conditions

60 A 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. Increased sensitivity accompanied by higher risk of background from endogenous biotin
B. Reduced sensitivity but faster protocol
C. No change in sensitivity but improved reproducibility
D. Lower background but reduced signal linearity
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