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?
To answer these questions, we need to use the information provided and perform some calculations. Let's break it down step by step.
1. To calculate the percentage purity of the LDH column fraction, we need to compare the optical density (OD) of the LDH band in the column fraction to the OD of the LDH standard band.
Given:
- OD of LDH band in column fraction = 42000 pixels
- OD of LDH standard band = 58000 pixels
To calculate percentage purity, we use the formula:
Percentage Purity = (OD of LDH band in column fraction / OD of LDH standard band) * 100
Percentage Purity = (42000 / 58000) * 100
Percentage Purity ≈ 72.41%
Therefore, the percentage purity of the LDH column fraction is approximately 72.41%.
2. To calculate the total amount of LDH in the column fraction, we need to use the total protein concentration of the column fraction and the volume.
Given:
- Total protein concentration of column fraction = 10 mg/ml
- Volume of column fraction = 2.4 ml
To calculate the total amount of LDH, we multiply the total protein concentration (mg/ml) by the volume (ml):
Total LDH = Total protein concentration * Volume
Total LDH = 10 mg/ml * 2.4 ml
Total LDH = 24 mg
Therefore, the total amount of LDH in the column fraction is 24 mg.