Decoding Publication Guidelines

Scientific publishers, funding agencies, and biotechnology and pharmaceutical companies are joining hands in an effort to improve accuracy and reproducibility of experimental data. To achieve this, journals are calling for good research practices, including sound experimental design, use of controls, and proper documentation of techniques.1,2,3,4,5

Take the Journal of Biological Chemistry2, for example. The Journal has spelled out specific requirements for authors, pertaining to analysis and submission of data from immunoblotting experiments. Among other guidelines, documentation of housekeeping antibody validation, normalization method, replicate samples, and statistical analysis is required. In order to help you successfully publish your data, we have taken a closer look at these guidelines and compiled resources to help you meet the documentation requirements.

The Journal of Biological Chemistry

Requires verification of quantitative data

“For studies reporting semi-quantitative analyses of immunoblots, authors should clearly explain how quantitative data were obtained, whether signal intensity has a linear relationship with antigen loading.2

Data quantitation has limitations

Quantitative Western blot (QWB) analysis is only accurate if the target protein and internal loading control can both be detected within the same linear range: a range that must be determined experimentally for each target and loading control.

LI-COR can help you establish the linear range of detection

Take a step by step approach to determine the linear range of detection for your target of interest and internal loading control of choice (total protein staining/housekeeping protein/signaling protein). The Linear Range Determination Protocol provides guidance on choosing loading volumes, measuring linear relationship between target and control signals, and accurate quantitation to help meet verification requirements.


The Journal of Biological Chemistry

Requires validation of housekeeping protein expression

“Housekeeping proteins should not be used for normalization without evidence that experimental manipulations do not affect their expression.2

This influences the accuracy of data normalization

With housekeeping protein (HKP) normalization, only one protein is employed for reference. The accuracy of the normalization procedure could be compromised by variations in the expression of the single HKP. HKPs affected by experimental conditions are not reliable tools for normalization, and will introduce error and may alter data analysis and interpretation.

LI-COR can help you validate your housekeeping protein expression before you use it to normalize

Fully characterize the expression of the housekeeping protein of your choice with LI-COR Housekeeping Protein Validation Protocol. If found stable, use the Housekeeping Protein Normalization Protocol for guidance on using validated HKP to normalize and quantitate Western blot data. Odyssey Loading Indicators will help assess consistency of sample loading across gel lanes.

If you find the expression of your HKP to vary with experimental conditions, consider REVERT Total Protein Stain for normalization of data to total protein loading.


The Journal of Biological Chemistry

Requires documentation of protein loading normalization

“For studies reporting semi-quantitative analyses of immunoblots, authors should clearly explain how protein loading was normalized among lanes.2

This affects experimental outcomes

Normalization is a critical part of attaining reproducible data from quantitative Western blots. Accuracy in quantitative immunoblotting relies on appropriate normalization and minimizing error.

LI-COR can help you choose the right normalization method for your experiment

Learn the foundational principles of normalization and review the most commonly used methods with LI-COR’s Western Blot Normalization Handbook. You will learn best practices for normalizing data using total protein staining, housekeeping proteins, and signaling proteins.

Once you have determined the appropriate method for your experiment, consider LI-COR reagent products for quantitative Western blotting: REVERT Total Protein Stain, antibodies against common housekeeping protein targets and Odyssey Loading Indicators.


The Journal of Biological Chemistry

Recommends use of total protein normalization

“Normalization of signal intensity to total protein loading is preferred.2

This is a different approach from widely used HKP normalization

With HKP normalization, only one protein is employed for reference, and the accuracy of the normalization procedure will be compromised by variations in the single HKP's expression.

LI-COR can help you normalize your data to total protein loading

Normalize your data to the entire amount of protein loaded in a lane, instead of a single HKP. The REVERT Total Protein Stain kit and the Total Protein Normalization Protocol will help you successfully generate accurate data for quantitative Western blotting.


The Journal of Biological Chemistry

Requires verification of signaling proteins

“Signals obtained using antibodies specific for phosphorylated epitopes should be normalized to the total protein level of the target protein.2

This affects data analysis and experimental results

Without normalizing signals from modified forms to total protein levels of the target protein, it is not possible to draw any conclusions about the effect of experimental conditions on post-translational modified forms of the protein.

LI-COR can help you accurately measure changes in signaling protein expression

Get a detailed protocol for using pan-specific antibodies as internal loading controls with Pan/Phospho Analysis for Western Blot Normalization. The protocol will guide you in analyzing changes in target protein phosphorylation, glycosylation, acetylation, ubiquitination, and other forms of post-translational modifications.


The Journal of Biological Chemistry

Requires documentation of replicates

“Authors must state the number of independent samples (biological replicates) and the number of replicate samples (technical replicates) and report how many times each experiment was repeated.2

Replicates improve reproducibility and accuracy of experimental findings

Replicate samples 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. Biological and technical replicates are both important, but each type of replicate addresses different questions.

LI-COR can help you integrate replicate samples in your study

Replication is an important part of Quantitative Western Blot analysis, and is used to confirm the validity of observed changes in protein levels. The Quantitative Western Blot Analysis with Replicates protocol will guide you in choosing and incorporating technical and biological replicates in your experimental design for reproducible data.


The Journal of Biological Chemistry

Requires publishing statistical analysis of experimental findings

“Statistical analyses of variation and precision for establishing differences between experimental groups should be preferably reported using the standard deviation (SD) or confidence intervals (CI).2

Statistical analyses are useful to interpret and validate results

Using Mean ± SEM bar graphs can be misleading and may alter the interpretation of data.

LI-COR can help you generate statistically significant data for quantitative Western blot analysis

Get detailed information on data interpretation, and calculation of coefficient of variation (% CV), arithmetic mean (average), and standard deviation for normalization and quantitative values. The Pan/Phospho Analysis for Western Blot Normalization, REVERT Total Protein Stain Normalization, and Housekeeping Protein Normalization protocols provide quantitative analysis support to help meet statistical analyses recommendations.


References

  1. Image Integrity. Nature. Macmillan Publishers Limited, 2016. Web. 31 July 2017.
  2. Instructions for Authors. The Journal of Biological Chemistry. American Society for Biochemistry and Molecular Biology. Web. 31 July 2017.
  3. Cell Press Digital Image Guidelines. Cell. CellPress. Web. 31 July 2017.
  4. Instructions for Authors. The Journal of Cell Biology. The Rockefeller University Press. Web. 31 July 2017.
  5. Instructions for preparing an initial manuscript. Science. American Association for the Advancement of Science. Web. 31 July 2017.
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