![]() ![]() Department of Commerce as a sanctioned and/or embargoed person or entity. (For a current list of all sanctioned and/or embargoed persons or entities, please consult the U.S. Department of Commerce).Īll rights to use the Bio-Rad product(s) are granted on condition that such rights are forfeited if You fail to comply with the terms of these Export Compliance Requirements.įor more information regarding U.S. Department of the Treasury, Office of Foreign Assets Control.Housekeeping Protein Normalization Protocol Introduction export laws and regulations, contact the U.S. ![]() In quantitative Western blotting (QWB), normalization mathematically corrects for unavoidable sample-to-sample and lane-to-lane variation by comparing the target protein to an internal loading control. Using a Housekeeping Protein (HKP) as an Internal Loading Control The internal loading control is used as an indicator of sample protein loading, to correct for loading variation and confirm that observed changes represent actual differences between samples.įor more normalization related resources, see " Further Reading". Housekeeping proteins (HKPs) are routinely used as loading controls for Western blot normalization. Accurate normalization requires stable expression of the HKP across all experimental conditions and treatments. Because HKP normalization relies on a single indicator of sample loading, variation in HKP expression leads to inconsistent estimation of sample loading and introduces experimental error that may alter data analysis.įor widely-used HKPs (such as actin, tubulin, and GAPDH), stable expression has generally been assumed. However, expression of common HKPs is now known to vary in response to certain experimental conditions, including cell confluence, disease state, drug treatment, and cell or tissue type.īefore an HKP is used for Western blot normalization, stable expression must be validated for the specific experimental context and treatments. For more information, see the Housekeeping Protein Validation Protocol ( /HKP-Validation LI-COR). This protocol describes how to use a housekeeping protein for Western blot normalization and quantitative analysis. This protocol is intended for use with near-infrared fluorescent Western blots. It also produces publishable figures which indicate exactly the quantification areas which is extremely important for a transparent measurement.Saturated bands and sample overloading frequently compromise the accuracy of QWB. I will try to get in contact with the authors and see if there might be a possibility to link it as plugin into ImageJ (e.g. It is freely available but not completely open source with access to the source code. The design of a quantitative western blot experimentģ.) Since I first tried to code a plugin which would combine the necessary functions for ImageJ, I stumbled over an existing and extremely good tool (also Java) with all the things you would need to run such an analysis (including calibration).Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS).Evaluating Strategies to Normalise Biological Replicates of Western Blot Data.A Defined Methodology for Reliable Quantification of Western Blot Data (Here you see the analysis methos indicated).Quantifying Western blots: Pitfalls of densitometry.So, here a few recommendations and very helpful links (since this is not my invention):ġ.) have a look at the following video seminar from Aldrin Gomes as an introduction to the problem on the experimental side:Ģ.) Here a few publications which help in getting a better understanding before the actial image analysis So, even while often suggested, drawing boxes around a band is not the proper way! 2: The ►Analyze ►Gel tools and the ►Analyze ►Calibrate… function provide theoretically everything what you would need but a clear guideline is still missing. That’s why I posted it here!Įverything I have found in the web so far was mostly going a in the wrong direction leading to non-reliable measurements! 1: Many students try to find a guideline for Western blot “quantification” online while using ImageJ. And I think the topic is important in the aspect of reliability and reproducibility in science. So the following topic is not directly linked to ImageJ necessarily but many of this community might still be interested in. Since once in a while the topic semi-quantitative Western blot analysis pops up and many people in biological sciences who do Western blots or gels are into that, I wanted to share some information on that. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |