Protein Analysis - Standard Curves Flashcards
Describe the key troubleshooting errors:
Low or no signal - ensure correct wavelength and check for protein degradation
High background noise - ensure plate reader is clean + remove any bubbles before reading
Non linear standard curve - ensure precise pipetting, use fresh reagents
Describe determining the R² Value of the Standard Curve:
Plot absorbance on y-axis vs concentration on x-axis
Generate a linear regression line y = mx + c
High R² = Strong linear relationship between absorbance and concentration
What are the controls used during a BCA ?
Negative control = buffer only (blank)
Positive control = known BSA standard
Describe the role of the Spectrophotometer in BCA Assay:
Measures the absorbance of the purple-coloured complex formed between copper ions and bicinchoninic acid (BCA) in the presence of proteins
The intensity of the purple colour is directly proportional to the protein concentration
Measures absorbance at 562 nm (where the BCA-copper complex absorbs light maximally)
Why are BCA Samples stored on ice ?
Prevents protein degradation - keeping samples on ice slows down enzymatic activity and prevents protein breakdown
Prevents denaturation as ice maintains protein stability
Reduces non specific reactions - low temperatures minimise background noise and unwanted chemical reactions during the assay
What is the equation of the line and what is represented by each component ?
Equation of a straight line is y = mx + c
Y = m x + c m is the gradient
Y = absorbance
X = concentration
M = slope
C = intercept
Describe the gold standard for pipetting:
Adjust the volume setting to the desired amount using the volume dial
Push the pipette firmly into the tip while rotating slightly until the tip is secure
Press the plunger to the first stop (slow and controlled) at a vertical angle
Immerse the tip 2–3 mm into the liquid (avoid touching the sides of the container)
Slowly release plunger to allow liquid to be drawn up ensuring no air bubbles are present and keeping pipette vertical during aspiration
Slowly press the plunger to the first stop to dispense the measured volume
Press to the second stop to expel any remaining liquid
Hold the plunger down and carefully lift the pipette tip out of the container
Discard the tip using the ejector button into a waste container
What are standards ?
Solutions or samples with known concentrations of the target analyte
Used to create standard curve for quantification
Describe the standard curve for BSA:
A graph used to determine the concentration of a unknown sample by comparing it known values
Typically generated by plotting absorbance or signal intensity against known concentrations of a standard
Used in assays like ELISA
BCA assays have a linear relationship between colorimetric absorption and protein concentration
What is BSA ?
Bicinchoninic acid assay/ Bradford assay
Both are variations of a a colorimetric method for measuring the amount of protein in a sample
In an alkaline solution, proteins reduce copper (II)(𝐶𝑢2+)to copper (I)(𝐶𝑢+)
BCA binds to copper (I) to form a purple complex
The greater the amount of protein, the greater the copper present, the deeper the purple colour
