1. Assume the concentration value of the control sample or the sample you need to normalize with as 100%.

2. Normalize the remaining test values with the control. Use the equation (100-((test1/control)*100)). Now your test values will be converted to percent change. Meaning test samples with protein higher than your control will have a negative percent value you need to reduce from your sample (eg: -20%) and test samples with lower concentrations of protein compared to your control will have a posetive percent value which you need to add to your sample (eg: +80%).

3. Fix volume of control you need to load according to the concentration. If your control has 2 ug/ul and you need to load 30 ugs, fix control volume as 15 ul.

4. Based on the percent difference, calculate how much test sample you need to load. If your test sample 1 has a change value of -20%. Load 12 ul of this sample. If test sample 2 has a value of +80%, load 27ul of it in the gel. (ie. (change value in percent * volume of control you need to load)+volume of control you need to load)

I used percentages to keep the concept simple. You can do this process using ratios or fold change values of 1. Excel or any spreadsheet software should make this work faster.

Hi, Mariah

If I understood correctly, you will load the samples/lysates directly in the well. Although many people do it, I prefer to normalize the samples before I load them.

If the samples you mentioned are already in sample buffer, you should proceed as Dr. Valissery said. If they are not yet in sample buffer, I would prefer to prepare the loading sample previously to applying them, but it's a personal choice.

Usually, I fix the concentration at 1ug/uL and calculate the amount of sample I need to reach a volume of 100 uL of ready-to-load sample in sample buffer. Working with tissue is easier, because it has higher protein concentrations, so it allows me to dilute the sample to 2ug/uL with lysis buffer and then again to 1 ug/uL with sample buffer, in order to have 1:1 dilution in the end.

So, in summary, I do this:

2ug/uL . 100 uL = [concentration of lysate] . [volume needed]

This dilution can be done with lysis buffer + protease/phosphatase inhibitor cocktail.

And then, I dilute it in half with sample buffer (50 uL sample at 2ug/uL + 50 uL 2x Laemmli buffer) to have a normalized 1 ug/uL sample ready to run.

As the sample buffer contains a lot of components, including SDS, reducing and denaturing agents, etc, I like to dilute the lysates 1:1 in sample buffer, as all the samples will have the same amount of each component.

Good luck!