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THE EFFECT OF SOLUBILIZATION ADDITIVES

In some cases the protein of interest could not be observed in the cell extracts, either with the
commercially available lysis products or with Aperto. A large number of additives were tested with
Aperto, at concentrations generally ranging from 12.5 50% final volume, and 5 additives were
found to have broad application. In the example below 4 of the 5 additives improve solubility
when compared to Aperto alone (A) and the competing product (C).
Examples requiring more stringent conditions are shown below
GROWTH CONDITONS
Growth conditions had a significant effect on expression and the ability to recover the difficult
proteins tested. Cells were typically grown in defined media then transferred to a modified Studier
Media for induction2. The following conditions were found to yield the best results.

FROM A FROZEN GLYCEROL STOCK

  1. 2% inoculum in Teknova Azure Media (Part number 3H5000) w/ 1% glucose and antibiotic
  2. Grow for 16 hrs at 20‐23C
  3. Transfer 25uls of this culture to a fresh 96 well plate containing 500uls of Azure Media 1% glucose + antibiotic
  4. Grow for 16 hrs at 20‐23C then raise temp to 37C and continue to grow for 3 hrs only (37C increases cell mass.....non inducing conditions)
  5. Transfer 25uls of this culture to a fresh 96 well plate containing 500uls Studier 2P Autoinduction Media (Part Number 3S2000)+ antibiotic and grow at 20‐23C for 16‐27 hrs

FROM A SINGLE COLONY ON A PLATE

  1. Prepare 500uls of Teknova Azure Media (Part number 3H5000) w/ 1% glucose and antibiotic in a 2ml deep well 96 well plate. Inoculate w/ a single colony from a fresh plate.
  2. Grow for 16 hrs at 20‐23C then raise temp to 37C and continue to grow for 3 hrs only (37C increases cell mass.....non inducing conditions)
  3. Transfer 25uls of this culture to a fresh 96 well plate containing 500uls Studier 2P Autoinduction Media (Part Number 3S2000) + antibiotic and grow at 20‐23C for 16‐27 hrs

References
1) Cells were generously provided by The Seattle Structural Genomics Center for Infectious Disease F.W. Studier (2005) Protein production by auto‐induction in high‐density shaking cultures. Prot. Exp. Pur. 41, 207‐234.

 

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