This week we have been used electrophoresis as a protein analysis tool. All you need to know about the polyacrylamide gel electrophoresis (PAGE) you can find in the manual or just google it:)

Before we start with this week report, I have one major comment. Please remember to put all the data in your lab reports, all the numbers, calculations, observation and so on. Do not hide any values or formulas you have used for calculations because I do not know where the data is coming from. That makes the lab report correcting very difficult what results in cutting the points (haha):). Remember about that.

So let's start. This week you have to estimate the protein size using the protein marker (ladder) as you reference. You have loaded the gel (run and staining was done for you) and you got your gel pictures from the blackboard.

This is an example of the Coomasie stained gel which I will use to show how to prepare the lab report.

Figure 1.

What you see here is small part of the bigger gel (only three wells). First lane is a ladder (protein mixture of known molecular weights, sizes indicated on the left side [kDa]), second and third lane represents an unknown protein samples which size we want to estimate. Proteins used here were purified (you can see that protein X has more contamitants than protein Y and that the amounts are different:). In your case one of the samples will be a crude bacterial extract (where you will see a lot of bands, what makes sense because there is a lot of different proteins in the cell - you do not have to estimate the size of all the bands, just comment on what you see) and the other one will be a purified protein, so you should expect only one band (size of this band you have to estimate).

First step is to measure the distance of the ladder bands from the origin (you take a ruller and go go go). What you do is what you see on the next figure:)

Figure 2.

You use the top and the bottom of the gel as your origin and stop point respectively (that do not matters a lot as it is a relative distance:) and you put your results in the table (I always try to measure the distance to the centre of the band but that is up to you. Remember only to do it exactly the same for each band)

Table 1. In the table you put the distances and you calculate the retention factor (Rf) by dividing the distance that a particular band overcomed by the stop point distance. For example for 20kDa band we have 11.90/13.08 = 0.595 (where in my case 13.08 is the stop point) . Then you calculate the Log10 of each Rf.

In the next step you draw a plot Ladder Size vs Log10(Rf) and you get this:

Using the linear regresion (that would be very accurate method), or by adding the trendline in the excell (to see how, go to the previous post on the 3rd year biochemistry practicals - Bradford) or manually (do not connect the points as you want, it is not kindergarden:), just make a straight line as close as possible to all the points on the plot) we obtain the standard curve. We will use this standard curve for our estimations.

Now we measure the distances of the bands of interest, calculate their Rf and Log10(Rf):

Then using the equation provided (or read manually) we estimate the molecular weight of your unknown samples.For example sample X:

y = -0.011x + 0.1016

where:

y - is your log10 of retention factor

x - is your molecular weight.

We know the retention factor so we are looking for the molecular weight. Using equation we have:

x = (y - 0.1016)/(-0.011) = (-0.66 - 0.1016)/(-0.011) = 69.2[kDa]

Molecular weight of the protein in the sample X estimated by aminoacid sequence is 69.323 (the sample X is the BSA - Bovine Serum Albumin protein). You can see that our result is very close to the real value.

You perform the analogous workflow for your lab report.

Again remember to put all the data!!!!

Have fun!!

CyA in two weeks.

Maciek

## No comments:

## Post a Comment