Western blot band anaylisis - (Jun/05/2012 )
Hi everyone, pls help me with the anaylisis of my western blot bands. The case is at follows, i must compare the expression of a certain protein X in control mice, sick mice, and mice resistant to the desease. For each type of mice i have 5 protein samples. With each sample (15 in total) i have made western blot so now i have the bands for my X protein for each sample, and also their respective loading control, actin. With these bands i put the image in the program image J and obtain the intensity of each band. Now i dont know how to proceed. In examples of how to analyze western blot bands they typically use 1 control and 1 experimental band, with wich they normalize the experimental bands to the control and the actin band, but i have 5 samples for control, so i dont know how to analyse my data, should i just take a mean of each group of samples? I need to do statistics later to see if it is significant the differential expression. Pls help me with the analysis!!
Ideally you would normalize each individual sample then aggregate the means for each group and do the comparisons on them.
First normalize your samples: for each sample so the ratio of the Protein X divided by the loading control.
Then do the average of the control group.
With the average, normalize every sample (even the controls) by dividing the value of the ratio you obtained in first place, by the average of the controls. The value you are obtaining is the fold change compared to the control group. To present the data, do the average of the double normalized ratio for each group. Note: The double normalized average of the control group should give you 1. Also you can obtain the standard error or deviation for each group.
I would apply an ANOVA to compare the different groups among themselves.
1- please measure protein conc. for each samples
2- load equal protein (micro gram), let controls, transfer to blot and develop for protein X, scan it
3- detect band intensity using image J, i.e calculate mean band intensity for each sample. < set the measurement to 8 bit, be sure that mean values are increasing with band intensity if there, otherwise invert image.
4- now again use your reference actin antibody to blot, scan and calculate mean value for all 5 sample
4- calculate avg of mean of protein X as you say / mean of reference protein for all 5 samples.
5- do this same for sick and resistance mouse samples.
6- now comparing these 3 values you would be able to see the change.