In WB, Should the Sample With the Lowest Gray Value Be Diluted for Equal Loading and Mixed With the Calculated Diluent?
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Original volume: 500 μL
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Final volume: 500 μL × 3 = 1500 μL
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Volume of diluent to add: 1500 μL − 500 μL = 1000 μL
The meaning of the dilution ratio calculated from the sample gray value is how many times the original sample needs to be diluted, rather than directly adding a volume of diluent equal to the ratio. Below is a procedure for diluting the sample according to the calculated dilution ratio:
Steps
1. Determine the target gray value: Select a target gray value, which is usually the one with the lowest gray value among the samples as the reference.
2. Calculate the dilution ratio: For each sample, calculate the ratio between its gray value and the target gray value. The formula is: dilution ratio = sample gray value / target gray value.
3. Perform dilution: According to the calculated dilution ratio, dilute each sample with the corresponding buffer solution.
Explanation of the Steps
Suppose you have a sample with a gray value of 200, and the target gray value is 100. According to the formula, dilution ratio = 200 / 100 = 2. This means you need to dilute the sample to half of its original concentration.
How to Dilute?
1. Calculate the final volume: If the sample is to be diluted to 2 times, then the final volume is twice the original volume.
2. Add diluent: If the original sample volume is 1 mL, you need to add another 1 mL of buffer solution, making the total volume 2 mL. In this way, the original 1 mL sample is diluted to 2 mL, i.e., diluted 2 times.
Example
If the original sample volume is 500 μL (microliters) and the dilution ratio is 3, this means you need to increase the final volume to 3 times the original volume:
Therefore, you need to add 1000 μL of diluent to the 500 μL sample to achieve a final volume of 1500 μL.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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