western blot quantification using imagej

#Western blot quantification using imagej manual
#Western blot quantification using imagej software

use a higher primary antibody dilution Īnd yes, you do need to go through this process for each antibody separately! 2.To fix any saturation problems and ensure you’re working within this range, you can then: The orange rectangle represents the linear range. This is where you want to work!įigure 1: The relationship between protein sample concentration and band intensity for a western blot. You should then find a linear line indicating where data is captured quantitatively- indicated by the orange box in Figure 1. To do this you need to serially dilute a known amount of your protein lysate, perform your Western, and plot the quantitated density of these Western blot bands (on the x-axis) against the amount you know you loaded (on the y-axis). To prevent saturation on film, you must empirically determine your linear range as shown in Figure 1.

#Western blot quantification using imagej manual

However, if your lab is a bit more old school and uses film to detect antibodies in a western blot, a more manual approach will be needed, as film can easily become saturated.

#Western blot quantification using imagej software

So take the time to formally review your software and see if this is the case. Luckily, many digital capturing systems come with software designed to detect saturation and automatically correct the exposure thereby ensuring your data analysis can be quantitative. If you are not working within the linear range, (i.e., if your detector or film can no longer absorb photons, it is saturated and you have hit your limit of detection) you are losing data. Find the Linear Rangeįor western blot quantification, you must ensure your image was captured in a manner sensitive enough to detect a change in what we call the “linear range”. Four Simple Steps for Western Blot Quantification 1. So on to the four critical steps for ensuring your (semi) quantification is accurate. This is because they cannot tell you exactly how much of your protein of interest there is, only how much there is relative to another sample.Įven though western blot quantification is only semi-quantitative, you still need to be rigorous in how you quantify your blots to ensure your results are as accurate as possible. Western Blots Are Semi-Quantitativeīefore we take you through the necessary steps for quantifying your western blots, we need to clarify that western blots are only semi-quantitative. This means you can see how the levels of your protein of interest change between time points, conditions, and treatments. The value you obtain is directly proportional to the concentration of your protein of interest.Ī quantitative western blot will allow you to measure relative changes between different conditions, so rather than just saying a protein is present or absent in a sample, you can go further and say your target protein is present x-times higher in one sample compared to another for example. Why Should You Perform Western Blot Quantification?įirst, what do we actually mean by western blot quantification? Put simply, this refers to the measurement of the signal emitted by your target protein. While it can be used to simply show the presence or absence of a protein in a sample, it can tell you so much more if you take the next step and perform western blot quantification.Īs scientists, we love nothing more than quantitative data! Here, we have pulled together the 4 key steps for you to perform western blot quantification. It separates proteins based on size and then uses antibodies to detect specific proteins within the sample. Western blotting is a commonly used lab technique to identify proteins within a sample.