I implemented a method for protein extraction of skin samples. I did not change the procedure but over time the protein yield decreased.
I homogenized skin biopsies using a bead mill homogenizer and Tris-HCl buffer with protease inhibitors and pH 7.6. Total protein was determined using a BCA Assay.
What influences do I need to consider that could cause the protein yield decrease?
If you do exactly the same procedure to exactly the same ingredients, you'd expect the same outcome. If the outcome changes, chances are that either the procedure or the ingredients have changed to cause this.
Perhaps you use a fresh batch of homogenisation buffer, that has a different composition from the old one? Perhaps you use the same buffer, but a component has gone off (that would result in a slow decline)? Perhaps the beads are different? You need to work on this systematically.
I’m sorry, did I get it right. The protein concentration in your homogenates decreases over time?
If so, you may need to increase the concentration of your protease inhibitor cocktail in your homogenates.
Sebastian Schmitt
, I did repeat the method with different samples and I measured the protein concentration of the centrifuged lysates (@10000g, 4°C, 10 min).First I got ~800 - 1000 µg/ml protein, now it is ~500 - 700 µg/ml.
Accuracy tests are in progress.
Eva-Maria Prugger Are the samples (skin) from different part of body? Do you expect the total protein amount will be similar among these samples? Were those skin samples collected at the same time? from the same individual?
It may depend on your method of protein determination and/or the presence of proteases that are insensitive to your protease inhibitor
Yuan-Yeu Yau , the samples are all from the same region but from different individuals. The samples are from 8 different time points, various biopsies at once. I expect a range of inter- and intra- suspect variation, but not this difference of protein yield between first and last individual.
Because of the differences in protein size, physical and chemical properties, and their tendency to bind and their vital activity
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