S-STEM Scholar Amanda Goodson's Blog: It's all about the gels this week.

After reading a journal article titled SDS Agarose Gels for Analysis of Proteins I discovered that they were using mini-casettes (8x8x0.1 cm) that were loaded with agarose gel. I have previously been using polyacrylamide mini-cassette gels but the concentration was not dense enough to capture the specific proteins that I am trying to capture. I had some of these mini-casettes in the refrigerator. I took them apart using the key (long flathead screwdriver), being very careful to only break the side seals in order to keep the seal at the bottom intact. I used liquid tape to secure the edges. 0.5 g of agarose was mixed with 50 mL of SDS on a hotplate. A 5 mL syringe was used to load gel. Unfortunately the liquid tape did not hold and the gel escaped enough to allow air bubbles into the cassette rendering it useless.
I decided to try a horizontal gel to see what kind of results I got. SDS (yelloe in color) was used to pour this gel (whitish-clear in color) and the wells were loaded with brain,urine and blood. A bio-marker was loaded but will only register proteins >6. After 88 minutes of electrophoresis at 100 volts I noticed that the gel was changing color. The gel was changing from yellow to clear as the proteins migrated from the negative pole to the positive pole. The positive reservoir was also changing from clear to yellow. When electrophoresis began both wells were clear from the buffer solution. I cut this gel in half and stained half of it with comassie blue and half of it with fast blast. The comassie blue showed banding but again, the bio-marker was not adequate so I still can not calculate the molecular weight of these proteins. The fast blast was too dark to visualize any banding.
I became extremely frustrated and decided to attempt another vertical gel. I was pleasantly surprised to see a package from Genscript waiting for me. Inside was PAGE mini-cassete gels with the concentration needed to capture the 4 kda proteins. Next I will run the PAGE and attempt to successfully pour an agarose vertical gel.