Decoding JBC Publication Guidelines

To help you successfully publish your quantitative Western blot data, we have taken a closer look at the Journal of Biological Chemistry2,3 (JBC) guidelines and compiled resources to help you meet the documentation requirements.

JBC has spelled out specific requirements for authors, pertaining to analysis and submission of data from immunoblotting experiments. Many other journals and grant agencies have detailed requirements as well, so it is always best to check the appropriate website and review the specific journals’ instructions for authors.

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.2,3

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 combined 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

LI-COR’s Combined Linear Range Determination Protocol provides guidance on choosing loading volumes, measuring linear relationship between target and control signals, determining the combined linear range, 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.2,3

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. If they are changing, this 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

LI-COR can help you determine if your housekeeping protein expression is changing under experimental conditions with the Housekeeping Protein Validation Protocol. If HKP expression is found to be stable, you can use the Housekeeping Protein Normalization Protocol to guide you in using validated HKP to normalize and quantitate Western blot data.


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.2,3

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.2,3

This is a different approach from widely-used HKP normalization

With HKP normalization, only one protein is employed for reference. 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

LI-COR can help you normalize your data to total protein loading with REVERT™ Total Protein Stain kit and the Total Protein Normalization Protocol.


The Journal of Biological Chemistry

Requires normalization of signaling proteins to total protein

“Signals obtained using antibodies specific for phosphorylated epitopes should be normalized to the total protein level of the target protein.2,3

This affects data analysis and experimental results

For making relative comparisons and to maximize accuracy, phospho-specific signals should be normalized against the total level of the target protein.

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

LI-COR can help you accurately measure changes in PTM protein as compared to total protein using the protocol for Pan/Phospho Analysis for Western Blot Normalization.


The Journal of Biological Chemistry

Requires inclusion and 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.2,3

Replicates improve reproducibility 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

LI-COR can help you understand why you need to include both types of replicates and how to integrate replicate samples in your study with the Quantitative Western Blot Analysis with Replicates.


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).2,3

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 in the analyses sections of our protocols.

With these products, protocols, and tools – and LI-COR’s +17 years of Western blotting experience – we can help you be successful regardless of where you are planning to publish your data.


Note: These resources should also assist you with other journals’ requirements but be sure to check guidelines for the journal to which you are submitting your publication.


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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. Collecting and Presenting Data.The Journal of Biological Chemistry. American Society for Biochemistry and Molecular Biology. Web. 9 May 2018.
  4. Cell Press Digital Image Guidelines. Cell. CellPress. Web. 31 July 2017.
  5. Instructions for Authors. The Journal of Cell Biology. The Rockefeller University Press. Web. 31 July 2017.
  6. Instructions for preparing an initial manuscript. Science. American Association for the Advancement of Science. Web. 31 July 2017.

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