Biology Reference
In-Depth Information
4
Notes
1. Alternatively membranes can be pelleted with a refrigerated
centrifuge at 50,000 ×
g
for 90 min at 4 °C
2. APS can be prepared as 10 % stock solution and kept as small
aliquots until use. Avoid multiple freeze-thawing.
3. Use concentrated HCl (12 N) and careful adjust the pH. Avoid
pH equalization after exceeding pH 6.8 with base.
4. Prepare directly before use. Be sure that the TMB is well solved
before the addition of the buffer.
5. If salt is added to fast, proteins can be denaturated.
6. A salt gradient (e.g., 30 %, 60 %, 90 %) can be used for a fur-
ther fractionation of the soluble proteins, if needed.
7. The volume depends on the size of the pellet and the used
fresh weight.
8. For quantifi cation of proteins a Bradford assay was used
(optional BCA, 660 nm (Pierce) or other methods can be
used).
9. First weight in the sucrose, KCl, phosphate buffer and water
and solve the sucrose then add dextran and PEG and mix
well. Weight precise, because all changes in the concentration
have an effect on the purity and yield of the plasma
membrane.
10. The lower phase is achieved by the addition of 9 g phase buffer
to the phase mixture, mixing the solution. The phase system is
kept at 4-8 °C over night. The upper phase is removed and the
lower phase is used for phase partitioning.
11. If the gel is casted from the top, the higher concentrated solu-
tion is fi lled in the mixing chamber and the lower concen-
trated solution in the reservoir chamber of the gradient
mixer.
12. The pH gradient can be varied by the use or combination of
different ampholytes.
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft
(DFG Lu 668/4-4) and the University of Hamburg (Young
Researcher Initiative grant to C.N. Meisrimler and PhD student
grant to D. Hopff).
