There are two independent questions. The first is how to quantify western blots, which can be done with various programs, and the second is how to do analyze them. The latter can become tricky depending on how the data is obtained. If all samples are obtained in a single run such that all data points are treated exactly the same, you can assume they are similar enough to do statistics. However, if they are obtained in separate runs, differences between the runs could affect the data. This could be a problem if the groups in your analysis correspond with separate runs, even when you have a proper loading control because you cannot assume that your target and your loading control are responding in the same way to small deviations in the protocol (like an hour extra overnight for the primary, the temperature of your transfer buffer, etc).

If you have properly obtained the data, you can use standard statistical tests like T-test of ANOVA to determine whether you have differences in the groups.

hi Adam,

first of all have you a software for image analysis available in the lab?

The main idea is to measure the density of your signal (bands) of interest. Then you normalize this signal with your loading control (actin,tubulin.... or total protein).

When you have this you compare your results with statistical test (depending the number of group that you have)

I don't know I'll ask what program there is to measure and compare densities of bands. I used storm scan software to scan the membrane.

Thanks

There are two independent questions. The first is how to quantify western blots, which can be done with various programs, and the second is how to do analyze them. The latter can become tricky depending on how the data is obtained. If all samples are obtained in a single run such that all data points are treated exactly the same, you can assume they are similar enough to do statistics. However, if they are obtained in separate runs, differences between the runs could affect the data. This could be a problem if the groups in your analysis correspond with separate runs, even when you have a proper loading control because you cannot assume that your target and your loading control are responding in the same way to small deviations in the protocol (like an hour extra overnight for the primary, the temperature of your transfer buffer, etc).

If you have properly obtained the data, you can use standard statistical tests like T-test of ANOVA to determine whether you have differences in the groups.

To add. if you have obtained data from different runs, but the two groups are included in each run, you can use an ANOVA with group as a fixed variable, and run as a random variable.

I completely agree with Kim van der Linde. It could be nice if you give us more details in your question if you want an efficient help from us :)

To add to above comments- yes, your image from the Storm is in a quantifiable format. You should have ImageQuant of some form that came with the scanner to analyze it - if not you can use imageJ. However, quantifying western blots can be inaccurate for several reasons---

- are all your samples in the linear range?

- were there any problems with transfer for certain bands?

- is the timing of acquisition exactly right?

- are all samples equally recognized by the antibody? (for instance, is there a change in the level of protein modification between samples and does that affect antibody recognition)

I'm sure there are more things too - just keep this in mind when you quantify your bands.

Thanks everyone ill let you know how I get on.

Http://rsbweb.nih.gov/ij/download.html