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Fig. 2 | BMC Biotechnology

Fig. 2

From: A hyper-thermostable α-amylase from Pyrococcus furiosus accumulates in Nicotiana tabacum as functional aggregates

Fig. 2

In native conditions, PFA forms aggregates. a. Zymogram (top panel) and western hybridization analysis with anti-PFA antibodies (lower panel) of plant-produced recombinant PFA from a stable transgenic plant on native PAGE. PBST: recombinant PFA extracted with PBST-based extraction buffer. Reducing: recombinant PFA extracted with reducing extraction buffer. μl: the amount of plant extract loaded onto each lane. Plant proteins were extracted from 20 mg of powdered freeze- dried whole leaf with 400 μl extraction buffer. Ten times more extract from PBST-based extraction was loaded onto the gel due to expected lower PFA extraction in this buffer. The zymogram does not show a difference in band intensities associated with loading volumes for either extraction method because the lowest loading volume contained enough PFA for digesting the starch present in the gel. b Crude extract in non-reducing buffer was filtered through a tangential flow filtration Pellicon XL cassette with 300 kDa cut-off membrane. lower functional PFA extraction in this buffer. c Crude extract in reducing buffer (80 ml) was sequentially filtered through Pellicon XL cassettes with 1000, 300, 100 and 10 kDa cut-off membranes. The membrane was washed with 20 ml buffer (wash). The permeate (Perm) and wash from one membrane were pooled and applied onto the next size cut-off membrane. The retentate (Ret) was about 8 ml in each case. Twenty μl of sample from each fraction were loaded onto each lane, and proteins were separated by SDS-PAGE

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