00:02
So you're looking at a sds page photo showing your lysate and the wash and elute after a his -tag protein that is being purified and also bind tightly to form an enzyme complex.
00:19
Based on the image, answer the following question.
00:22
What is the size in keta of the purified protein? so if you look at the lysate, there are two very prominent bands.
00:31
They're together.
00:34
So most likely, they are being recognized by the antibody and they are the his -tag protein.
00:47
And this is between 25 to 37 kilodalton.
00:52
Judging by the position of the band, it should be closer to 37.
00:57
So let's say the two band is about 32 kilodalton.
01:00
So this is judging by the letter.
01:04
So the letter will give you the approximate size of the band.
01:11
We call it protein letter.
01:13
This is in the first column or what we call the first well.
01:18
Now you can see which band is approximately what size.
01:23
But judging by the position, our two band is at around 32 kilodalton.
01:28
The second question, what do the smear of the bands in the lysate represent? so lysate has a lot of protein.
01:35
So the two most prominent bands are most likely the his -tag protein that is being recognized specifically.
02:03
So this is why it's prominent.
02:10
However, when you overload the sample to the lane, you might see non -specific bands, which are smears.
02:37
So this non -specific protein bands are protein that has similar sequence compared to the target protein, but they are also recognized by the antibody non -specifically due to the overload of the sample.
03:01
So when you have a lot of protein in that lane, and then they are tend to be recognized by the antibody non -specifically because a lot of proteins share common domains.
03:13
So the antibody recognize the target protein may also recognize protein with similar structure.
03:24
And so they are being recognized by the antibody by mistake.
03:30
So these are the smear front.
03:31
So again, this is caused by the overload of the sample.
03:34
If you reduce the protein loaded in that lane, you can reduce the smear just like the wash and elute lanes...