Question

You find yourself setting up another Western blot, however. today you are preparing the samples to load onto the SDS-PAGE gel. You are using a 4-20%SDS-PAGE gel that can hold a maximum of 30 μしper 1 You remember from you so decided to prepare each sample with a final volume of 25 uL. However, no one is perfect at pipetting, so make sure each sample is made at 1.5X the total volume needed per well. The protein you are interested in lowly expressed in your cells, therefore, you need to make sure each sample has a final pro of 50 ug. With the information provided, complete the chart below. (16 points) awesome Cell Biology lab that you should never load the maximum i into each well tein concentration Protein concentration is protein .Lof 3540 ug/mL HL protein TL of water -μL 4x buffer 31.5 4315 4.375 4315 3150 ug/mL 2975 ㎍/mL 31.5 31s Before the prepared samples from question 7 are loaded onto the SDS-PAGE gel, we must boil the sample for 5 minutes. Why are the samples boiled? And why does this boiling take place in the sample buffer? (6 points) 8

Answer 1

Q7)

Protein concentration	µl protein sample	µl of water	µl 4X sample buffer
3540 µg/ml	14.12	18.68	4.70
3150 µg/ml	15.87	16.34	5.29
2975 µg/ml	16.80	13.10	5.60

Explanation:

3540 µg/ml = 3540 µg/ 1000 µl is the concentration. 50 µg is the required protein in each well. So protein sample is 50* 1000/3540 = 14.12 µl.

Now, 4X sample buffer means 1 part of buffer in 3 parts of protein sample. So 4X sample buffer required is 14.12/3 = 4.70 µl.

As the required volume per well is 37.5 µl so the water required will be: 37.5-14.12-4.70 = 18.68.

Same is the calculation for other rows.

Q8)

Samples are boiled for 5 minutes to denature the proteins. But it should be kept in mind that longer duration could lead to aggregation of proteins.

Samples must be boiled in sample buffers as proteins are dissociated to polypeptides (their subunits) and a negative charge is imparted to these subunits. Disulfide bonds are also broken with the help of sample buffer. Hence, denaturing is enhanced.