Measuring a protein by Western blotting

A Western blot measures how much of one particular protein a tissue contains. The proteins are extracted from the tissue, their total concentration is measured so that the same amount can be loaded for every sample, and they are separated by size by SDS polyacrylamide gel electrophoresis (SDS-PAGE). The detergent SDS unfolds the proteins and gives them negative charge in proportion to their length, so that in the gel they move towards the positive electrode and are sieved by size; the discontinuous gel system most laboratories use was described by Laemmli (Laemmli, 1970).

The separated proteins are then moved by an electric current out of the gel and onto a membrane, which holds them in place. The free surface of the membrane is blocked with excess protein, and the protein of interest is detected with a specific antibody followed by a second, labelled antibody. Towbin and colleagues introduced this electrophoretic transfer to nitrocellulose and detected as little as 100 pg of protein with a peroxidase reaction (Towbin et al., 1979).

Protein concentration is commonly measured with the bicinchoninic acid (BCA) assay, whose purple colour increases with protein concentration over a broad range and which tolerates nonionic detergents and simple buffer salts better than the older Lowry method (Smith et al., 1985). Today the bands are usually made visible by chemiluminescence, light produced by the enzyme on the second antibody, and recorded with a digital camera; their intensities are then measured (densitometry) and corrected with a loading control.

The target is Gx, a hypothetical protein of approximately 55 kDa, in mouse hippocampus. The simulator records the true amount of Gx in each mouse, so the decisions that changed the result are shown at the end.

Learning objectives

• Design a blot with the mouse as the unit of analysis, enough biological replicates, coded samples and randomised lane order
• Lyse tissue with protease inhibitors, run a BCA assay, choose the dilution and the standard curve, and calculate a loading table
• Choose a gel for the size of the protein, and build a transfer sandwich in the right orientation, knowing why proteins move towards the anode
• Choose blocking, antibodies and a loading control, and recognise the signs of a cross-reactive antibody, degradation and a failed transfer
• Choose an exposure that avoids saturation, and quantify bands with equal boxes and local background subtraction
• Compare groups with Welch's t-test, a 95% confidence interval and an effect size, and interpret the result honestly

Ethics and safety

The tissue comes from mice used under a protocol approved by an Institutional Animal Ethics Committee; in India such work is governed by the CCSEA under the Prevention of Cruelty to Animals Act 1960, with equivalent frameworks in the European Union (Directive 2010/63/EU), the United States (Animal Welfare Act and PHS Policy) and the United Kingdom (Animals (Scientific Procedures) Act 1986). Group sizes are justified in advance and kept to the minimum that answers the question, and tissue from one set of animals can often serve several assays (Reduction, one of the 3Rs). Experiments are reported following the ARRIVE guidelines, developed by the NC3Rs in the United Kingdom and endorsed by journals internationally (Percie du Sert et al., 2020), and many journals also ask for the full, uncropped image of every blot. At the bench, lysis buffers, acrylamide (toxic before it has polymerised), SDS, methanol in the transfer buffer and the high voltages of electrophoresis require laboratory safety training, gloves and eye protection; power supplies are switched off before lids are removed.

Key terms

• Lysate: The liquid obtained when tissue is broken up in a detergent buffer and the debris is removed by centrifugation; it contains the soluble proteins.
• Protease inhibitors: Chemicals added to the lysis buffer that block the enzymes (proteases) which would otherwise digest the proteins once the cells are broken.
• BCA assay: A colour test for total protein: protein reduces copper ions, and bicinchoninic acid binds the reduced copper to give a purple colour read at 562 nm (A562).
• Standard curve: Absorbance measured for samples of known concentration (here albumin), used to convert the absorbance of an unknown sample into a concentration.
• SDS-PAGE: Electrophoresis in a polyacrylamide gel after the detergent SDS has unfolded the proteins and given them negative charge, so that they separate by size.
• kDa: Kilodalton, the unit of molecular weight used for proteins; read the size of a band against the molecular-weight marker.
• Anode and cathode: The positive (anode, red lead) and negative (cathode, black lead) electrodes. SDS-coated proteins move towards the anode, in the gel and during transfer.
• Transfer sandwich: Sponge, filter paper, gel, membrane, filter paper, sponge, pressed together in a cassette with the membrane on the anode side of the gel.
• Ponceau S: A red, reversible stain for all proteins on the membrane, used to check the transfer and as a total-protein loading control.
• Blocking: Covering the free surface of the membrane with protein (here 5% milk) so that antibodies bind only to their target.
• Primary and secondary antibody: The primary antibody binds the protein of interest; the secondary binds the primary and carries the enzyme that produces the light.
• Chemiluminescence: Light produced by the enzyme peroxidase acting on a substrate; it is recorded by a camera during the chosen exposure.
• Saturation: A camera pixel that has reached its maximum value (65,535 for a 16-bit camera) records no further light, so a saturated band is underestimated.
• Loading control: A measure of the protein in each lane (total protein, or a housekeeping protein) by which the band of interest is divided.