Begin by using Schirmer strip fragments to collect the tear. Place these strips in a 600-microliter microcentrifuge tube containing 20 microliters of sterile water with a protease inhibitor and centrifuge at 600g for one hour at four degrees Celsius. Using a 7.5-centimeter stainless steel injector needle, puncture the tip of the 600-microliter tube containing the sample.
Transfer this punctured tube to a new sterilized 1.5-milliliter tube and centrifuge the samples at 12, 000g for 10 minutes at four degrees Celsius to recover the volume of diluted tears. Combine the supernatant recovered from all the samples to create a protein pool of approximately 200 microliters. Heat the protein samples at 99 degrees Celsius for four minutes to denature the proteins.
Vortex the samples briefly to mix the components. Then, load 30 micrograms of quantified protein content from each sample onto a 10%SDS-PAGE gel and run the samples at 90 volts for two hours using a standard method. After electrophoresis, cover the gel with staining solution and incubate for 30 minutes to stain the proteins.
Then, rinse the gel with the destaining solution several times until the background becomes clear and protein bands are visible. Acquire images with a gel documentation system. Coomassie stained SDS-PAGE analysis of total protein extracts revealed patterns of protein expression in total retina and tears.
