00:01
You're studying a protein that i think consists of a heterodimer consistent composed of 30 and 40 kiloliters subunit.
00:10
So they're joined together by disulfide bond.
00:13
So in addition to your protein sample, you have available a sds -page gel, a gel electrophoresis apparatus, and a bio -red precision protein standard.
00:26
And you also have access to two different buffers.
00:29
A has 4 % sds and 5 % beta mecapital ethanol.
00:45
So beta me stands for the beta mecapital ethanol.
00:51
And b contains 4 % sds but no beta mecapital ethanol.
01:03
Now as per the practical, samples are heated to 95 degrees celsius for four minutes before they load it to sds -page.
01:14
And then you can do electrophoresis.
01:17
So the question asks you to describe the experimental method that would allow you to test your hypothesis.
01:22
Now provide sufficient details such as the interest reader could faithfully repeat your experiment.
01:29
Also describe the chemical and biochemical involved in a treatment.
01:36
So basically the idea is that we have a protein complex.
01:41
One subunit is 30 kilodalton, the other is slightly bigger, 40.
01:51
And they are linked by disulfide bond.
01:57
So disulfide bond is a covalent bond that formed between a specific amino acid called cysteine.
02:05
So it's a covalent bond that means if we treat the complex with sds, sds is going to break all the non -covalent interactions such as the hydrogen bond, the ionic interaction, and hydrophobic interaction.
02:26
However, sds is not able to break down the disulfide bond.
02:32
Now the disulfide bond can only be broken by the beta mecapital ethanol.
02:37
And this is why we include it in one of the buffer.
02:41
So our idea, basically the biochemical theory behind it is that sds can't break down disulfide bond, but beta mecapital ethanol can.
03:33
So beta mecapital ethanol is a reducing agent.
03:41
The reducing agent is going to break the disulfide bond.
03:46
It's a redox reaction.
03:48
So now this is the biochemical background about it.
03:52
Let's design experiment.
03:54
Step one, you are going to have protein sample.
04:05
You can split them into two.
04:09
So they are actually the same thing.
04:12
So both contain this protein.
04:29
Second, you add buffer a to sample one and add buffer b to sample two.
04:58
And then of course, you heat up the sample at 95 degree for four minutes before they load it to sds page.
05:26
Obviously, the next step is sds page.
05:31
So you load sample.
05:35
Well, first of all, you have to load the protein ladder, the bio -red precision protein standard.
05:46
Lane one, bio -red protein standard, probably about five micrometer to make sure that you have enough protein in the standard...
