You have purifiedLDH using affinity chromatography. Using the bradford assay you determined the total protein concentration of the column fraction to be 10 mg/ml in a final volume of 2.4 ml. You diluted some of your column fraction to 1 mg/ml using SDS Page sample loading buffer and loaded 15 ul on to an SDS page gel. You also loaded 10 ul of a 1mg/ml LDH standard in an adjacent lane. The stained sds page gel revealed that your ldh column fraction contained a number of bands besides ldh. Upon scanning you found that the ldh band in your cokumn fraction had an optical density of 42000 pixels. The optical density of the ldh standard band was 58000 pixels.1. What is the percentage purity of your ldh column fraction?2. How many total mg of ldh are there in your column fraction?

Question

You have purifiedLDH using affinity chromatography. Using the bradford assay you determined the total protein concentration of the column fraction to be 10 mg/ml in a final volume of 2.4 ml. You diluted some of your column fraction to 1 mg/ml using SDS Page sample loading buffer and loaded 15 ul on to an SDS page gel. You also loaded 10 ul of a 1mg/ml LDH standard in an adjacent lane. The stained sds page gel revealed that your ldh column fraction contained a number of bands besides ldh. Upon scanning you found that the ldh band in your cokumn fraction had an optical density of 42000 pixels. The optical density of the ldh standard band was 58000 pixels.1. What is the percentage purity of your ldh column fraction?2. How many total mg of ldh are there in your column fraction?

Answer 1

To determine the percentage purity of your LDH column fraction, you can compare the optical densities of the LDH band in your fraction and the LDH standard band. Here's how you can calculate it:

1. Calculate the percentage purity:
- Optical density of LDH band in your column fraction: 42000 pixels
- Optical density of LDH standard band: 58000 pixels

Percentage purity = (Optical density of LDH band in your column fraction / Optical density of LDH standard band) × 100
Percentage purity = (42000 / 58000) × 100
Percentage purity ≈ 72.41%

So, the percentage purity of your LDH column fraction is approximately 72.41%.

Now, let's calculate the total amount of LDH in your column fraction:

2. Calculate the total mass of LDH in the 15 μl of your column fraction:
- Protein concentration of the column fraction: 10 mg/ml
- Volume of the column fraction loaded: 15 μl

Mass of LDH in the loaded volume = Protein concentration × Volume
Mass of LDH in the loaded volume = 10 mg/ml × 15 μl
Mass of LDH in the loaded volume = 150 μg

To find the total amount of LDH in the column fraction, we need to account for the dilution factor used when preparing the sample.

- Dilution factor = Protein concentration of diluted sample / Protein concentration of the column fraction
- Dilution factor = 1 mg/ml / 10 mg/ml
- Dilution factor = 0.1

Total mass of LDH in the column fraction = Mass of LDH in the loaded volume / Dilution factor
Total mass of LDH in the column fraction = 150 μg / 0.1
Total mass of LDH in the column fraction = 1500 μg (or 1.5 mg)

Therefore, the total amount of LDH in your column fraction is 1.5 mg.