Question

I need to calculate efficiency of IP.For that, I had 300microliter lysates containing eGFP (300microliter). I used 2 microliter to carry out BCA assay and find concentration which turned out to be 3309ng/microliter.I froze 10 microgram of the lysates (3 microliter) because it has total protein (this will be analyzed on western blot later).

The remaining of the lysates (295 microliter) I used to carry out IP to isolate eGFP. In the end I have eGFP in elution buffer (35 microliters).
Now I ran both IP extract (25 microliters) and all of the frozen total protein (add 7 microliter buffer to make loading easy) on Western Blot.
I have two lanes (total protein and eGFP) with known intensities. How can I find IP efficiency?

Answer 1

To calculate IP efficiency, the Western Blot band intensities for eGFP from the IP extract and total protein are compared. This qualitative measure, which ideally should be made quantitative with a standard curve and loading controls, provides the proportion of eGFP protein that was pulled down relative to the amount present in the lysate.

Step-by-step explanation:

Calculating Immunoprecipitation (IP) Efficiency

To calculate the efficiency of IP, first, we need to establish the amount of eGFP protein in the total lysate and in the IP eluate. You mentioned that you ran both the IP extract (25 microliters) and all of the frozen total protein (10 micrograms in 10 microliters Lysates + 7 microliters buffer) on a Western Blot. By comparing the band intensities on the blot for these two samples, you can get a relative measure of how much of the eGFP protein was pulled down by the IP compared to how much was present in the original lysate.

However, it's important to note that for a quantitative comparison, you need consistent loading controls and ideally, a standard curve of eGFP to ensure that your measurements are within a linear range. Assuming you have those, you would calculate the proportion of the intensity of the band from the IP extract relative to the total protein lane intensity. That proportion, when corrected for the different volumes loaded (even if you loaded all of the IP, you need to consider the concentration differences), will give you the apparent efficiency of your IP.

In practice, this might look something like calculating the mass of eGFP in each band (using a known standard curve), and then the efficiency percentage would be (mass of eGFP in IP band) / (mass of eGFP in total lysate band) * 100, adjusted for any differences in sample volumes and concentration.