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- Prewarmed LB Agar plates: Dissolve 1 % (w/v) tryptone,
0.5 % (w/v) yeast extract, 1 % (w/v) NaCl, and 1.5 % (w/v)
microagar in ddH 2 O and sterilize by autoclaving. Allow the
mixture to cool to around 50 °C before adding 25 μg/mL
kanamycin. Gently mix by swirling and pour a thin layer
(~10 mL and 5 mm thick) into a sterile petri dish. Allow the
plates to cool completely before storing upside down at 4 °C
and prewarm to room temperature before use. - Prewarmed SOC medium: 2 % (w/v) tryptone, 0.5 % (w/v)
yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgSO 4 ,
10 mM MgCl 2 , 20 mM glucose prepared in ddH 2 O and fi lter-
sterilized. Store at 4 °C and prewarm to room temperature
before use. - Auto induction medium: Add 1 L ( see Note 5 ) of ddH 2 O and
10 mL of glycerol to 60 g Overnight Express Instant TB
medium (Novagen) and stir using a magnetic stirrer until the
medium is dissolved. Microwave to sterilize and allow cooling
to at least 50 °C before adding kanamycin to 25 μg/mL. This
medium can be stored at 4 °C and should be prewarmed to
room temperature before use.
The Novex NuPAGE SDS-PAGE Gel System (Invitrogen) is
described here and while any SDS-PAGE system would suffi ce the
percentage of the gels and the chosen running buffer are impor-
tant for different parts of the analysis.- Mini gel system for SDS-PAGE analysis (XCell Surelock,
Invitrogen). - SDS-PAGE gels (10 % and 4–12 %) (NuPAGE Bis-Tris Gels,
Invitrogen). - MOPS and MES SDS running buffers (NuPage 20× solution,
Invitrogen) diluted to a 1× working solution using ddH 2 O
(MOPS and MES Buffers, respectively). - Coomassie protein strain for SDS-PAGE gels (SimplyBlue
SafeStain, Invitrogen). - Sample buffer (NuPAGE LDS Sample Buffer (4×), Invitrogen).
- Sample reducing agent (NuPAGE Reducing Agent (10×),
Invitrogen). - General reducing agent (NuPAGE antioxidant, Invitrogen).
- Protein molecular weight markers (SeeBlue Plus2, Invitrogen).
- Microcentrifuge tubes.
All purifi cation buffers should be fi ltered (0.2 μm) before use and
additionally buffers for size exclusion chromatography (gel fi ltra-
tion) should be degassed for around 20 min.2.2 SDS-PAGE
Analysis
2.3 Protein
Purifi cation
Karen McLuskey et al.http://www.ebook3000.com