How do biologists separate, identify and quantify molecules and cells in the laboratory?
Laboratory techniques for biologists: dilutions and standard curves, separation by centrifugation, chromatography and electrophoresis, antibody techniques (immunoassay, ELISA, blotting), aseptic technique, cell culture and cell counting.
An SQA Advanced Higher Biology answer on laboratory techniques, covering linear and log dilution series and standard curves, separation by centrifugation, chromatography and gel electrophoresis, antibody techniques including immunoassay and blotting, aseptic technique and cell culture, and counting cells with a haemocytometer.
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What this key area is asking
The SQA wants you to describe the core laboratory techniques a research biologist uses: how to make and use dilution series and standard curves, how to separate substances by centrifugation, chromatography and electrophoresis, how antibodies are used to detect and quantify specific molecules, and how cells are cultured aseptically and counted. This is the practical toolkit that underpins the whole of Cells and Proteins.
Dilutions and standard curves
Many measurements start by preparing solutions of known concentration.
To measure how much of a coloured (or colour-producing) substance is present, a colorimeter or spectrophotometer measures absorbance. The instrument is first set to zero with a blank, a tube containing everything except the substance of interest, so that only the substance being measured contributes to the reading.
Separation techniques
Three techniques separate the components of a mixture on different principles.
Chromatography separates substances by their relative solubility and affinity for a stationary phase and a mobile phase. A substance that is more soluble in the mobile phase, and binds the stationary phase weakly, travels further. Paper, thin-layer and column chromatography all use this principle.
Antibody techniques
Antibodies bind one specific antigen, which makes them ideal detection tools.
Aseptic technique and cell culture
Working with living cells means keeping out unwanted microorganisms.
Microorganisms are grown in liquid broth or on solid agar, while animal and plant cells are grown as cell cultures that need a sterile medium, the right temperature and added growth factors. A cell line is a culture that can keep dividing.
Examples in context
Example 1. Subcellular fractionation. To study mitochondria, researchers break open cells in a cold, buffered, isotonic solution and use differential centrifugation. Low speeds pellet nuclei; higher speeds pellet mitochondria, giving a clean fraction to study respiration. This shows centrifugation separating organelles by density.
Example 2. A pregnancy test as an immunoassay. A home pregnancy test uses antibodies that bind the hormone hCG. When hCG is present, the antibody-bound complex produces a visible line, exactly the antigen-antibody detection that underlies ELISA and other immunoassays, but read by eye rather than a plate reader.
Try this
Q1. State the principle by which gel electrophoresis separates molecules. [1 mark]
- Cue. By charge and size as they move through a gel towards an electrode.
Q2. Explain why a blank is used before measuring with a colorimeter. [2 marks]
- Cue. It zeroes the instrument so only the substance of interest contributes to the absorbance reading.
Exam-style practice questions
Practice questions written in the style of SQA exam questions on this dot point, with worked answer explainers. The year tag is the paper they imitate, not the source.
SQA AH style4 marksDescribe how a standard curve is produced and used to find the concentration of an unknown solution.Show worked answer →
A 4-mark answer needs the preparation, the measurement and the use of the curve.
A series of standards of known concentration is prepared, usually by a dilution series from a stock solution. The absorbance of each standard is measured in a colorimeter or spectrophotometer, after first zeroing the instrument with a blank that contains everything except the substance being measured.
Absorbance is then plotted against known concentration to give the standard curve. The unknown solution is measured under the same conditions, and its absorbance is read across to the curve to find its concentration by interpolation.
Markers reward (1) standards of known concentration, (2) use of a blank to calibrate, (3) plot of absorbance against concentration, and (4) reading the unknown off the curve.
SQA AH style3 marksExplain why proteins are commonly separated by gel electrophoresis according to size.Show worked answer →
A 3-mark answer needs charge, the gel and the size effect.
The protein sample is treated with a detergent that coats every protein with a uniform negative charge, so the proteins move through the gel towards the positive electrode at a rate that depends on size rather than their own charge.
The gel acts as a molecular sieve: small molecules pass through the pores of the gel more easily and so travel further, while large molecules are held back and travel a shorter distance.
Markers reward (1) proteins given a uniform charge, (2) movement towards the positive electrode through a gel that acts as a sieve, and (3) small molecules travelling further than large ones.
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Sources & how we know this
- SQA Advanced Higher Biology Course Specification — SQA (2019)