Protein Electrophoresis Samples Preparation 1. In tube 1, add 10 ?l of prestaine
ID: 260685 • Letter: P
Question
Protein Electrophoresis
Samples Preparation
1. In tube 1, add 10 ?l of prestained protein ladder standards
2. In tube 2, take the 10 ?l of the first protein sample (that has been prepared for you) and add 5 ?L of 5x dye.
3. In tube 3, take the 10 ?l of the second protein sample (that has been prepared for you) and add 5 ?L of 5x dye.
4. In tube 4, take 2.5 ?l of the second protein sample (that has been prepared for you) and add 5 ?L of 5x dye.
5. Heat the tubes 2 through 4 at 95 ° C for 5 min just before loading the sample into the gel.
Questions
1. You loaded the same sample onto two lanes of your gel but at different concentrations.
A. Compare and contrast the results in those two lanes.
B. When may you want to load more sample? When may you want to load less sample?
Explanation / Answer
Answer A. The results of the lane no. 3 and 4 in which sample sample has been loaded with different concentrations will result in a band pattern in which the resolved protein bands will be more prominent and visible in the case of lane no. 3 as the amount of protein loaded in lane no. 3 is more. On the other hand the visibility of proteins and the band size will be low in lane no. 4 as less protein is loaded. If we proceed for western blotting in these samples after running SDS page then some proteins which are low in expression may not be detectable in this sample as the amount of protein is low and it may not cross the threshold level of detection by ECL method but may be detectable in lane 3 as the amount of protein loaded is more and it may cross the threshold levels.
Answer B. We may want to load more samples if we are looking for a protein that has very low expression in the system from which the sample has been isolated (cell line, tissue etc.). If the level of expression of a particular protein is low then more sample is needed to be loaded on the gel so as it crosses the threshold levels for detection by ECL method. Likewise we will load less sample if the expression level of protein is high because loading more sample in this case may lead to the non specific binding of antibodies and false positive detection of bands or non specific banding pattern.
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