The Half Decent Pharmaceutical Chemistry Blog

Summer is rapidly approaching and, apart from the warmer and sunnier days, if you’re a man, there’s a chance you have realised it by the sudden change in the diets of many women around you. Haven’t you noticed your girlfriend doesn’t you to take her out for dinner any more? Have you checked the ratio Diet Coke : Beer in her fridge? Haven’t most of your female colleagues at work started to sit around the table (where you have lunch all together) to eat, unlike you, carrots and yoghurt? Well, they are terrified, thinking about the day YOU will innocently propose to go the beach and they will have to squeeze themselves inside a microscopic bikini (perhaps, on that particular occasion they’d opt for a more forgiving swimming costume, pretending that it’s still too cold for them). Trust me, she hates you because you REALLY don’t care about your funny skin colour (after months in the lab, who could be bronzed?) and how fat you are.

Another common theme for a conversation among your female lab-mates (if you ever bothered listening) is the diet to try not to arrive…unprepared for the first Saturday at the beach. Magazines and tabloids are full of  last-minute, radical diets. Needless to say, most of these tips are for desperate, running out of time people and have no scientific rationale behind. So, they are useless and often dangerous.

Here at The Half Decent Pharmaceutical Chemistry Blog, we like to do things properly and, even when it comes to diet tips, we want to be taken seriously and put science behind what we say. So, for the diet I’m going to unveil for all the ladies reading this, I want you to believe me when I say I’ve tried it.

I’ve indeed what I call the Lowry diet on Wednesday and Thursday. In a few words, you skip the lunch break because you are in the middle of a protein extraction for a Western Blot: I know you’ll be thinking you should be in no hurry with the protease inhibitors and sodium orthovanadate and fluoride you have suspended your extract in. Don’t fool yourself, please: we all know you should be quick at boiling your samples and quantifying it (this, merely, because you’re curious to know how many microlitres you’ll have to load on your gel).
Whatever the reason, if you harvest at, say, midday (because it’s 24 hours after you performed a transfection), you’ll be right in the middle of your experiment, perhaps preparing your BSA standards for the calibration curve.
The Lowry assay, in fact, is a popular, maybe old-fashioned, certainly reliable and sensitive way of quantifying the concentration of proteins. It basically consists of two parts: in the former step you perform a Biuret reaction, which means you add an alkaline solution of Cu2+, that reacts with peptide bonds yielding Cu+ ions. Subsequently, Folin-Ciocalteau reagent is added: this mixture of phosphomolybdate and phosphotungstate undergoes a reduction to heteromolybdenum blue, which is coupled with the oxidation of aromatic amino acids in the presence of copper ions.

To sum up, the solution turns blue (picture to come soon, psi*psi) with an intensity that depends on the amount of tyrosine and trytophan and, therefore, the overall amount of proteins present in the cuvette. The precise concentration is determined by comparing the absorbance of the sample at 750 nm with those of standard solutions of BSA, used to draw a calibration curve.

This said, you generally need to wait 15 minutes after the addition of each of the two solutions, so you’ll be too busy to have lunch. It definitely works!