WorksheetsProteomics
Total questions: 48
Worksheet time: 1hrs 27mins
What is the advantage of using recombinant DNA technology in protein production?
precise manipulation of DNA sequences
expression of proteins in various host organisms
perfect recombinant DNA every time
Decreased antibiotic resistance
What is the purpose of expressing a protein as a fusion construct containing an epitope tag?
to express the protein at high levels
to denature the protein
to purify the protein
to create an inter-membrane protein
What type of affinity tags are commonly used in IMAC?
GFP
Glutathione S-transferase-tag
poly-adenine tails
His-tag
What does IMAC stand for?
What is the basic principle behind IMAC?
what is the significance/function of Nickel in the Ni-NTA resin?
what is the difference between native and non-native protein purification?
Native protein purification can be exploited to -- proteins such as enzyme subunits and binding proteins.
(a)
What is needed for successful his-tag protein purification under native conditions
Presence of inclusion bodies
Solubility of the 6xHis-tagged protein
Denaturation of the protein
Binding to the Ni-NTA resin only
Which factor contributes to nonspecific binding of proteins in native conditions during affinity purification?
Presence of inclusion bodies
pH near the isoelectric point
Denaturation of the protein
Low concentration of imidazole in buffers
How can we reduce non specific binding when performing IMAC batch purification?
Including low concentrations of imidazole in the binding buffer.
Conducting purification under denaturing conditions
Using high concentrations of imidazole
Reducing pH in the wash buffer
washing lysate using low salt concentrations
Which method is recommended for elution of 6xHis-tagged proteins when protein damage by pH reduction is a concern?
Increasing imidazole concentration
Decreasing pH to 4.5–5.3
Adding chelating agents like EDTA
Using step-wise pH gradients
Why do we equilibrate the columns before adding the cell lysate?
between prokaryotic (bacterial) and eukaryotic expression systems, which requires more stringent protein purification methods and why?
how can we wash non-tagged protein from the column(i.e what goes into wash buffers to wash away non-tagged proteins)?
What determines the elution pH of monomeric 6xHis-tagged proteins during affinity purification?
pH 4.5
pH 7.0
pH 6.0
pH 5.9
what does pKa mean and why is it important for determining protein elution?
Which parameter should be adjusted for optimization of binding in batch purification when the concentration of 6xHis-tagged proteins is low?
pH
Temperature
Salt concentration
imidazole concentration
list and describe the 4 methods of eluting tagged proteins
What is the binding capacity of Ni-NTA resins typically for 6xHis-tagged proteins?
1–2 mg/ml
20–30 mg/ml
5–10 mg/ml
50–100 mg/ml
Which method is recommended to elute 6xHis-tagged proteins when the presence of metal ions in the eluate is undesirable?
EDTA chelation
Increasing pH to 9
Using high concentrations of imidazole
Decreasing pH to 3
What should be adjusted if the 6xHis-tagged protein does not bind during affinity purification using a 10x concentrated cell lysate with 10 mM imidazole?
Imidazole
Salt concentration
concentration
Temperature
pH
what is the principle behind ion exchange chromatography?
Discuss the importance of purification under native conditions for 6xHis-tagged proteins and the strategies employed to ensure successful purification, including the role of solubility and the potential copurification of associated proteins.
Compare and contrast the batch and column purification procedures for Ni-NTA affinity chromatography, highlighting their respective advantages and applications in protein purification.
Evaluate the methods used to reduce nonspecific binding of proteins during affinity purification on Ni-NTA resins under native conditions, including the use of low imidazole concentrations, adjustment of pH, and optimization of binding conditions.
Critically analyze the elution methods for 6xHis-tagged proteins from Ni-NTA resins, comparing the effectiveness and appropriateness of imidazole elution, pH reduction, and chelating agents, considering factors such as protein stability and metal ion contamination
What properties are used to separate proteins in column chromatography?
Isoelectric point and solubility
Molecular weight and shape
Hydrodynamic radius, electric charge, and hydrophobicity
Amino acid composition
Discuss the principles of ion exchange chromatography and how it separates molecules
Compare and contrast the modes of ion exchange chromatography: cation exchange chromatography and anion exchange chromatography.
Explain the factors that influence the binding and elution of molecules in ion exchange chromatography
How does pH affect the ion exchange chromatography process?
Evaluate the importance of understanding the isoelectric point of a target protein in designing effective ion exchange chromatography methods, considering its implications for protein binding and elution behavior
Discuss the importance of salt gradients in ion exchange chromatography and their impact on the elution of target molecules
How can ion exchange chromatography be coupled with other chromatographic techniques or analytical methods for enhanced purification and analysis? Provide examples
Evaluate the steps involved in desalting a protein sample for subsequent ion exchange chromatography, including the rationale behind dilution and the use of specific buffer conditions.
Explain the principle behind Immunoprecipitation.
Which commercial example of magnetic beads is commonly used in immunoprecipitation experiments?
Magnetic nanoparticles
Potato beads
Agarose beads
Sepharose beads
In immunoprecipitation using magnetic beads, what is Protein A (or G) used for?
How can the Scribble protein be eluted from the magnetic bead complex?
Which region of the IgG antibody includes the protein target binding region?
Constant region
Fc region
Fab region
Variable region
In immunoprecipitation using magnetic beads, what is used to preserve interactions with Scribble binding proteins during washing?
High pH buffer
Low pH buffer
Strong detergents
Gentle washes
What does trypsin do?
why is trypsin digestion optimal when preparing samples for LC-MS/MS?
explain the principle of SDS-PAGE electrophoresis
What role does polyacrylamide gel play in SDS-PAGE analysis?
It separates proteins based on mass
It maintains protein structure
It binds to SDS and proteins
It neutralizes the charge of proteins
--is typically used to stain proteins on the polyacrylamide gel after electrophoresis
(a)
How are protein samples prepared before loading onto the gel in SDS-PAGE analysis?
They are diluted with water
They are treated with reducing agents
They are treated with protease enzymes
They are heated and cooled rapidly
