Replicates improve the reproducibility and accuracy of experimental findings. They are important because they confirm the validity of observed changes in protein levels. Without replication, it is impossible to know if an effect is real or simply an artifact of experimental noise or variation, which can directly affect conclusions made about experimental findings.
There are two types of replicates: biological and technical. Each type addresses different questions1,2,3. Peer-reviewed journals, such as the Journal of Biological Chemistry, have specific guidelines in regards to replicates.
“Authors must state the number of independent samples (biological replications) and the number of replicate samples (technical replicates) and report how many times each experiment was repeated.”
— Instructions for Authors. The Journal of Biological Chemistry
Technical vs. Biological Replicates: Which Do You Need to Include?
Technical replicates are repeated measurements used to establish the variability of a protocol or assay, and determine if an experimental effect is large enough to be reliably distinguished from the assay noise1. Examples may include loading multiple lanes with each sample on the same blot, running multiple blots in parallel, or repeating the blot with the same samples on different days.
Figure 1. Technical replicates help identify variation in technique. For example, lysate derived from a mouse and treated under a set of experimental conditions (A, B, C), then run and measured independently three times, will help identify variation in technique.
Technical replicates evaluate the precision and reproducibility of an assay, to determine if the observed effect can be reliably measured. When technical replicates are highly variable, it is more difficult to separate the observed effect from the assay variation. You may need to identify and reduce sources of error in your protocol to increase the precision of your assay.
Technical replicates do not address the biological relevance of the results.
Biological replicates are parallel measurements of biologically distinct and independently generated samples, used to control for biological variation and determine if the experimental effect is biologically relevant. The effect should be reproducibly observed in independent biological samples. Demonstration of a similar effect in another biological context or system can provide further confirmation. Examples include analysis of samples from multiple mice rather than a single mouse, or from multiple batches of independently cultured and treated cells.
Figure 2. Biological replicates derived from independent samples help capture random biological variation. For example, lysates derived from 3 mice and treated under the same set of experimental conditions (A, B, C), will help identify variation resulting from the biology.
To demonstrate the same effect in a different experimental context, the experiment might be repeated in multiple cell lines, in related cell types or tissues, or with other biological systems.
An appropriate replication strategy should be developed for each experimental context. Several recent papers discuss considerations for choosing technical and biological replicates1,2,3.
This protocol, Quantitative Western Blot Analysis with Replicates, will guide you in choosing and incorporating technical and biological replicates in your experimental design for reproducible data. It includes calculations for replicate analysis as well as how to interpret the data you obtain.
Additional Resources to Help You Get the Best Data
LI-COR has additional resources that you can use as you plan your quantitative Western blot strategy.
- Determining the Linear Range for Quantitative Western Blot Detection provides guidelines for characterizing the linear range of detection for your system.
- Housekeeping Protein Validation Protocol walks you through the steps of how to validate that the expression of an HKP is constant across all samples and unaffected by the specific experimental context and conditions. If expression varies, then the HKP cannot be used for normalization.
- Housekeeping Protein Normalization Protocol shows you how to use housekeeping proteins for Western blot normalization, as long as you have validated that their expression does not change under your experimental conditions.
- REVERT™ Total Protein Stain Normalization Protocol describes how to use REVERT Total Protein Stain for Western blot normalization and quantitative analysis, fast becoming the gold standard for normalization of protein loading.
- Pan/Phospho Analysis for Western Blot Normalization provides guidelines on how to use total and post-translationally modified proteins for Western blot normalization.
- Naegle K, Gough NR, Yaffe MB. Criteria for biological reproducibility: what does “n” mean? Sci Signal. 8 (371): fs7 (2015).
- Blainey P, Krzywinski M, Altman N. Replication: quality is often more important than quantity. Nat Meth. 11(9): 879-80 (2014).
- Vaux DL, Fidler F, Cumming G. Replicates and repeats – what is the difference and is it significant? EMBO reports 13(4): 291-96 (2012).