2,695
edits
Lidstrom: SDS-PAGE: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
→Questions/Answers/Facts
| Line 286: | Line 286: | ||
==Questions/Answers/Facts== | ==Questions/Answers/Facts== | ||
===How do you prepare soluble and insoluble fractions for SDS-PAGE?=== | |||
Yakov Kipnis of the Baker lab wrote in 11/2014: | |||
"Soluble and insoluble fractions of cell lysate look distinct on gel (ratios of band intensities for various proteins is quite different), therefore if you see large portion of unlysed cells contaminating insoluble fraction distinction between soluble/insoluble becomes less pronounced. To make the comparison easier I typically run 3 lanes/sample (total cell lysate/soluble(sup after spin)/insoluble(pellet after spin) I always try to load similar amount of protein on a gel. For "total" and "soluble" it is straightforward, to help solubilize as much of "insoluble" as possible I resuspend pellet in small volume of 8-9 M urea (avoid GuHCl it destroys SDS gels) first and reconstitute to the same volume before taking small aliquot for gel sample. | |||
Example: total volume of the lysate 500 uL (take 10 uL for gel="total"), spin (take 10 uL="soluble"), remove supernatant as much as possible, add 20-50 uL 9M urea, resuspend pellet, dilute to 490 uL by water/buffer (take 10 uL="insoluble"). This way theoretically overlay of "soluble" + "insoluble" lanes should give you "total", if corresponding bands do not sum up there is a chance something unaccounted happened. Later you can skip "total" to save space on gel." | |||
===What do all of the reagents do? How long do they last? === | ===What do all of the reagents do? How long do they last? === | ||
* SDS | * SDS | ||
