Week 3 events

Hi everyone!!!

I hope that you guys are doing OK. =)

This week i had a long Wednesday in the Lab... It is LSM2201A again.....My groupmates and I were having slow progress on our experiments that we finished at 7pm...Yikes... Our TA was quite nice to stay with us to supervise our experiment where he could have ate dinner at 5pm.=) But, generally it was quite enjoyable and fruitful day..I think i have gained more confidence in using the micro-pipette =)

Our experiment firstly were to isolate and purify lactate dehydrogenase from rat liver. In case that “lactate dehydrogenase” sounds like Greek to you, it is the enzyme that catalyzes the reaction from pyruvate to lactate in anaerobic respiration . Generally, the reason why you have sore muscles after running is due to the formation of lactate in your muscles. As you run, your muscles will utilize anaerobic respiration to fulfill their oxygen debt. Lactate is formed and as it decreases the pH inside the muscles, it becomes toxic, resulting in pain.

We got the buffers we prepared a fortnight ago from the freezer. What is a buffer? A buffer is basically a solution that has a weak acid/base with its conjugate base/acid. The pH will change very little when a small amt of strong acid or base is added to it. Buffers have a wide variety of applications. The chemistry of buffers can be summed up in the Henderson-Hasselbalch equation: ph = pKa + log [A/HA]

As most biochemical processes have low tolerance to extreme pH, buffers are common in living organisms. For example, the hydrogen carbonate and carbonic acid one in blood. In the biochemical lab, one common buffer is tris(hydroxymethyl)aminomethane, affectionally known as “tris”. This is a major component in the buffers we use.

We put the liver extract by affinity chromatography through a column. Affinity chromatography is a method of separating biological mixtures in a lab on the basis of a reversible interaction between a protein and a ligand (which has a similar structure to a substrate and a cofactor) in an immobile phase, which is usually a form of a gel.. This form of separation has several uses like the purification of an antibody in an blood serum. Whereas the protein we want is trapped to the gel, the other proteins will not be trapped. After we remove the other proteins, we can wash out our desired protein using another buffer. This process is usually termed elution. In our experiemnt, we use the blue gel called affi -gel blue. The dye has a similar structure to NADH and NAD, which allows the dye to bind to a wide variety of proteins. There was a problem with the rat liver extract we were originally given which were too lumpy which clogged up our column, as a result we couldn't get our fractions. We had our 12 fractions later when we were given more homogenized liver extract.

We set out to find how much enzyme activity and how much the protein we had in our fractions to figure out which fraction had the most enzyme. I worked on the enzyme activity assay while my lab mate worked on the Protein assay. I used a spectrophotometer which is an instrument that measures intensity as a function of light or color. Basically it measures the transmittance of light between a reference solution and a test solution. Light from the source lamp is diffracted to a series of individual wavelengths of light. Light of a single frequency is then passed through a sample. This intensity is measured with a light sensor and the transmittance is compared with the transmission through a reference sample. Before we start the machine, we have to zero the sample to set a baseline in which all other readings are compared relative to the zeroed sample. Usually the machine would give a reading of % absorbance. Spectrophotometers have a wide variety of uses, and in this case by monitoring the change in absorbacne over time, we can measure the activity of the enzyme..cool huh..

When we were finished with our experiments, we handed over the samples to our TA and went back finally to home. Whew!!! A long day for us and the TA too!!!=)