10 Questions
What is the main difference between direct and indirect detection in ELISA?
In direct detection, the antibody specific to the antigen has the enzyme, while in indirect detection, a second antibody with the enzyme binds to the primary antibody.
How is specific activity of an enzyme calculated?
Specific activity is calculated by normalizing the total activity to the total protein amount.
What is the purpose of a standard curve in western/immunoblot detection?
The standard curve is used to establish a relationship between protein concentration and signal intensity.
What is the purpose of staining proteins with Coomassie blue?
To determine the success of protein purification and detect proteins
What is the significance of the limit of detection of a nanogram in protein detection?
It indicates the sensitivity of the protein detection method
How does isoelectric focusing separate proteins based on their properties?
By separating proteins based on their isoelectric points under non-denaturing conditions
What is the purpose of 2D PAGE in protein detection?
To separate proteins based on both size and isoelectric points
What is the significance of measuring absorbance at 260 and 280 nm to detect DNA or RNA in a sample?
To determine if the sample contains DNA, RNA, or protein based on the A260/280 ratio.
What challenges arise during the amino acid determination process, and how are they addressed?
Challenges include deamidation and conversion of cysteine derivatives. Deamidation is addressed by designating the total population as Asx and Glx, while cysteine conversion is addressed by protecting it with performic acid to create cystic acid.
How is the abundance of each amino acid detected, and why is the reaction with PITC important?
The abundance of each amino acid is detected by reacting with PITC under slightly alkaline conditions. The reaction with PITC is important because it forms the PTC-AA structure, helping in amino acid detection.
Study Notes
Detection Methods in ELISA
- Direct detection involves direct binding of an antibody to the target antigen, whereas indirect detection involves binding of a primary antibody to the target antigen and a secondary antibody to the primary antibody.
Enzyme Specific Activity
- Specific activity of an enzyme is calculated by measuring the amount of substrate converted per unit of enzyme per unit of time, usually expressed as units per milligram of protein.
Western/Immunoblot Detection
- A standard curve is used to quantify the amount of protein in a sample by comparing the signal intensity to a known concentration of the protein.
Protein Staining
- Coomassie blue staining is used to visualize proteins in a sample, allowing for the detection of protein bands and estimation of protein molecular weight.
Protein Detection
- A limit of detection of a nanogram in protein detection is significant because it allows for the detection of very small amounts of protein, which is important in many biological and medical applications.
Isoelectric Focusing
- Isoelectric focusing separates proteins based on their isoelectric point (pI), which is the pH at which the protein has no net charge, by using a pH gradient to focus proteins at their pI.
2D PAGE
- 2D PAGE (Polyacrylamide Gel Electrophoresis) separates proteins based on two properties: size and charge, allowing for the separation of complex protein mixtures.
Nucleic Acid Detection
- Measuring absorbance at 260 and 280 nm allows for the detection of DNA or RNA in a sample, as these wavelengths correspond to the absorbance maxima of nucleic acids.
Amino Acid Determination
- Challenges arise during amino acid determination due to factors such as sample contamination, instrument variability, and chemical interference, which can be addressed through the use of quality control measures, instrument calibration, and chemical optimization.
Amino Acid Detection
- The abundance of each amino acid is detected through the reaction with PITC (Phenylisothiocyanate), which forms a stable derivative that can be analyzed by HPLC or other techniques, allowing for the detection of specific amino acids in a sample.
This quiz covers the principles of ELISA (Enzyme-Linked Immunosorbent Assay) for detecting specific proteins, including the direct and indirect detection methods. It explains the process of coating the surface with sample antigens, blocking unoccupied sites, and using specific antibodies and enzymes to produce a colored product upon binding.
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