quantifications of western blots with imagej

QUANTIFICATIONS OF WESTERN BLOTS WITH IMAGEJ HOW TO

The following are some factors that can cause signals to fall outside the linear range of the system. Normalization accuracy could be affected. The measured signal for ILC and target will not vary to the same degree with sample loading, so the Core Normalization principle will not hold. Outside the linear range of sample protein loading, the relationship between the amount of protein and the measured signal is unknown. The span of signal intensities that display a linear relationship between amount of protein on the membrane and signal intensity recorded by the detector. More details on each method and additional information about normalization is provided later in the handbook. The necessary validation will vary based on the method you choose. The flow chart on the following page will walk you through the steps necessary to choose an appropriate normalization strategy for your experiment. Quick Stop for Choosing a Normalization Strategy In this illustration of signal detected on a 'real' blot, the target protein varies and the total protein varies as well. Further, detection method limitations may affect the accuracy of detection. Even if it were possible to get the same amount of protein from each sample into a gel, transfer efficiency from the gel to the blot will be different from lane to lane. However, in the real world, it is not possible to load exactly the same amount of protein from each sample. Although the target protein expression may vary, the same total protein was loaded for each sample. This illustration shows signal from an ideal Western blot. The signal for your target protein might be the same in each sample, or it might differ based on your experimental treatment. Each sample would contain a uniform, consistent amount of sample protein, and the total protein signal measured for each sample would be the same. Under ideal circumstances, normalization would not be necessary for Western blots. To quickly get started with choosing a normalization method, see 'Quick Stop for Choosing a Normalization Strategy'. To learn more about the key principles of normalization, see 'Normalization Basics'. The reader must apply the general information from this guide to specific protocols as appropriate. This guide is a general overview of normalization principles, not a specific protocol. Throughout this guide, the Journal of Biological Chemistry's submission guidelines and other peer reviewed normalization method papers will be referenced to provide context and sources of additional information.

QUANTIFICATIONS OF WESTERN BLOTS WITH IMAGEJ HOW TO

This guide summarizes the basic principles of normalization and explains how to use these principles to accurately normalize Western blot data using one of three different normalization strategies. Recent changes, notably updates to the Journal of Biological Chemistry's submission guidelines (1), necessitate a closer look at traditionally accepted normalization practices. Normalization is a critical part of attaining reproducible data from quantitative Western blots.

Copy the results into an excel file and analyse as necessary.Normalization Handbook What You Will Learn from This Guide.Be sure to count the peaks on the plot as to determine which peak corresponds to the peak of interest.

Go to the results window and the areas are calculated in numerical order.

Click on selected peaks (all peaks which are above the baseline) in order.

Select peaks using the wand (tracing) tool.

Isolate individual peaks which protrude significantly from the baseline. Use the line tool to connect the peaks to the baseline as they would if the peaks were individual and not connected in a line.

If necessary use the line tool to connect peaks to the baseline.

The baseline is to remove the 'noise' from the background of the scanned gel.

Plot lane by selecting Analyze->Gels->Plot Lanes or press CTRL-3.

Using side arrow key, slide box over to next lane and select Analyse->Gels>Select Next Lane or press CTRL-2.

Select lane either by selecting Analyze->Gels->Select First Lane or press CTRL-1.

Using a rectangular box (box tool)select entire lane.

Save gel image and adjust to be vertically oriented.

Quantifications of western blots with imagej