Further Questions- Expt 4

There are three stages in the experiment. Firstly, Isolation. Secondly, Purification and lastly, Analysis.

In this post, I'm gonna talk bout the Analysis of this experiment which is also the results and discussion.

I could remember clearly what happened this day. The spectrophotometer we were using in our lab was down! I was quite happy for a moment because I thought that we do not have to discuss about the results. BUT, duh we had other working ones in other rooms. Ha Ha.

So, we went to other lab room and used the spectrophotometer to find out our absorbance readings of our samples! The spectrophotometer was set at 476nm ( the wavelength at which GFP strongly absorbs and gives out its usual fluroscence.)

RESULTS!

Our group's absorbance readings:


(click on the image for clearer view)



The Graph.... (click on the image for clearer view)



Further questions/ Discussion

1)
Plot the graph of your A476 absorbance values (Y-axis) vs fraction number. Comment on your chromtogram.

Theory wise, we can deduce that the higher the concentration of the absorbing substance, the greater the amount of light will be absorbed from the green flourescent protein (GFP). From our graph above, we can see that fraction number 2 has the highest absorbance value. This meant that fraction 2 has the highest amount of GFP. The absorbance value for the first fraction is low as it contained mostly ammonium bicarbonate that we used for our control. This is because as we first added in the sample, the sample takes time to travel down the column and so the first fraction would show little GFP. After the first fraction, the gradient of the graph started to rise sharply. This shows that the GFP is starting to elute till the third fraction where the absorbance value reaches the peak. When it reaches the peak, it meant that most of the GFP has been eluted. From then on, the gradient starts to fall as it will contain lesser GFP thus decrease in absorbance value.


2)
Comparing a protein of Mr 50000Kd and GFP having a Mr of about 27000kD, the protein with a higher molecular weight will be eluted first. In this case, the protein of Mr 50000kD will be eluted after GFP.

This is because semi-permeable resin has a range of pore sizes that determines the size of proteins that can be separated with the column. This is called the fractionation range or exclusion range of the resin.

Proteins larger than the exclusion range of the resin are unable to enter the pores and pass quickly through the column in the spaces between the resin. Small proteins and other low molecular weight substances that are below the exclusion range of the resin enter all the pores in the resin and their movement through the column is slowed because they must pass through the entire volume of the column. Proteins of a size that falls within the exclusion range of the column will enter only a portion of the pores. The movement of these proteins will be slowed according to their size; smaller proteins will move through the column more slowly because they must pass through a larger volume.