![]() Naturally each lane will have a slightly different amount of protein in it therefore by normalising to expression of stably expressed “housekeeping” proteins such as GAPDH or β-actin this can help to ensure that any changes in expression of a target protein are not just due to differences in loading. Next it is critical to normalise the signal intensity for protein loading.This step is easily achieved in both bespoke analysis software and freeware such as ImageJ ( ). Differences in background across the blot may not be consistent leading to changes in band darkness that are influenced by background, not signal intensity. It is next important to subtract any background noise from the image.Above each datapoint is a representative illustration of the band seen. Reducing the concentration of primary antibody is also another way of reducing saturation.įigure 2: Example curve showing the saturation of western blot signal at high levels of bound protein. one is always saturated to see the other). However, where there are large differences in protein abundance between samples then it may be necessary to re-attempt the western blot with more similar protein concentrations if it isn’t possible to have both bands properly exposed (i.e. However, saturation is a relatively easy problem to solve as by reducing the exposure time this can be avoided. If the signal is saturated then it isn’t possible to accurately compare different protein abundances. Before trying to take any quantitative measurements it is critical to make sure that the signal is not saturated and is in its linear range (figure 2).Crucially this technique can only inform of relative changes in abundance between samples therefore without known standards, cannot be used to give exact concentrations. Protein expression can be quantitatively compared within a immunoblot using densitometry. How to quantify protein expression from a Western blot A high R2 value indicates increased accuracy in interpretation of the unknown proteins MW. Example standard curve of log(MW) against relative migration (Rf). The actual protein molecular weight as determined by mass spectrometry is likely to differ from the estimated through western blotting due to differences in glycosylation status, experimental inaccuracies and some proteins not being amenable to full denaturation by SDS.įigure 1.The general equation is: which in figure 1 simplifies to: Use the equation of the best fit line to calculate the mass of the target using its Rf.This should be linear if the samples are fully denatured and the gel percentage was adequate for sufficient separation. Plot the log(MW) of the protein standards against relative migration distance (Rf) on a graph and generate a curve of best fit.This can either be measured using a ruler or within appropriate software. Calculate the relative migration distance (Rf) of each protein standard and the target protein using the equation below.Run the gel using a molecular weight ladder, transfer to the membrane and then visualise the proteins using a dye such as Coomassie blue or Ponceau.The molecular weight of a protein can be estimated by comparing the migration of proteins of known molecular weight (such as in a protein ladder) and the target protein. Measuring the molecular weight of proteins Western blots are essential for comparing protein expression between experimental conditions and for working out the molecular weight of proteins. Copy the results into an excel file and analyse as necessary.Western blots are a key technique where antibodies are used to probe immobilised proteins on a membrane in order to visualise them.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. ![]()
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