. Author manuscript; available in PMC: 2023 Aug 10.

Published in final edited form as: J Vis Exp. 2021 Sep 10;(175):10.3791/63012. doi: 10.3791/63012

Table 2. Reagents for preparing S30A and S30B wash buffers, adapted from Kieser et al ⁴⁵.

Reagent	Concentration	Amount	Additional Notes
HEPES	1 M	0.1 L	Adjust pH to 7.5 with 5M KOH
MgCl₂	1 M	0.1 L
NH₄Cl	4 M	0.5 L	Takes time to dissolve (endothermic). Use a stir bar and let equilibrate to room temperature.
ddH₂O for S30A	55.5 M	730 mL	For S30A.
ddH₂O for S30B	55.5 M	927.5 mL	ForS30B.
DTT	1 M	5 mL	Make fresh on the day of use and keep on ice.
The above reagents are required to prepare 1L of the S30A and S30B buffers. Dissolve all components fully in ddH₂O. Autoclave separately. We recommend using a pen to mark the side of container (Duran flask)
S30A buffer
On the day of use, add the following in order. Keep on ice.
Component	Stock concentration	Volume	Final concentration
Deionized water	55.5 M	730 mL
HEPES	1 M	10 mL	10 mM
MgCl₂	1 M	10 mL	10 mM
NH₄Cl	4 M	250 mL	1 M
DTT	1 M	2 mL	2 mM
S30B buffer
On the day of use, add the following in order. Keep on ice.
Component	Stock concentration	Volume	Final concentration
ddH₂O for S30B	55.5 M	927.5 mL
HEPES	1 M	50 mL	50 mM
MgCl₂	1 M	10 mL	10 mM
NH₄Cl	4 M	12.5 mL	50 mM
DTT	1 M	2 mL	2 mM

Table 2. Reagents for preparing S30A and S30B wash buffers, adapted from Kieser et al 45.