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--- purification:tev:faq [2016/07/13 15:24] – bonnefon
+++ purification:tev:faq [2023/11/01 20:19] (current) – external edit 127.0.0.1
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 ====== TEV Protease FAQ ======
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 === What protease inhibitors are known not to affect TEV? ===
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 === What are typical reaction conditions? ===
-The "standard" reaction buffer for TEV protease is 50 mM Tris-HCl (pH 8.0),  0.5 mM EDTA and 1mM DTT. The duration of the cleavage reaction is typically overnight, although lots of cleavage will happen in the first few hours and prolonged incubation times may not lead to proportional increases in cleavage. TEV protease is maximally active at 34 °C, but we recommend performing the digest at room temperature (20 °C) or 4 °C. TEV protease is only three-fold less active at 4 °C than at 20 °C [Nallamsetty et al., 2004].
+The "standard" reaction buffer for TEV protease is 50 mM Tris-HCl (pH 8.0),  0.5 mM EDTA and 1mM DTT. The duration of the cleavage reaction is typically overnight, although lots of cleavage will happen in the first few hours and prolonged incubation times may not lead to proportional increases in cleavage. TEV protease is maximally active at 34 °C, but we recommend performing the digest at room temperature (20 °C) or 4 °C. TEV protease is only three-fold less active at 4 °C than at 20 °C [[http://www.sciencedirect.com/science/article/pii/S104659280400302X|(Nallamsetty et al., 2004)]].
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 === Can I do an on-column cleavage? ===
-On-column cleavage is possible but comparatively inefficient. TEV protease is not especially stable and has a tendency to bind nonspecifically to column materials. The addition of “stabilizers” like sorbitol, glycerol, or salt may be helpful. For an example of on-column cleavage by immobilized TEV protease, see Puhl et al. (2009).
+On-column cleavage is possible but comparatively inefficient. TEV protease is not especially stable and has a tendency to bind nonspecifically to column materials. The addition of “stabilizers” like sorbitol, glycerol, or salt may be helpful. For an example of on-column cleavage by immobilized TEV protease [[http://onlinelibrary.wiley.com/wol1/doi/10.1042/BA20080063/full|(Puhl et al., 2009)]].
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 === How sensitive is TEV protease to other additives and reaction parameters? ===
-TEV protease has a relatively flat activity profile at pH values between 4 and 9. It is 50% as active in 0.5 M NaCl as it is in the absence of salt [Nallamsetty et al., 2004]. TEV protease is maximally active at 34 °C, but we recommend performing digests at room temperature (20 °C) or 4 °C. The activity of TEV protease is approximately 3-fold greater at 20 °C than at 4 °C [Nallamsetty et al., 2004]. It will tolerate a range of buffers, including phosphate, MES, and acetate. TEV protease is not adversely affected by the addition of glycerol or sorbitol (up to at least 40% w/v). TEV protease is sensitive to some detergents [Mohanty et al.,
+TEV protease has a relatively flat activity profile at pH values between 4 and 9. It is 50% as active in 0.5 M NaCl as it is in the absence of salt [Nallamsetty et al., 2004]. TEV protease is maximally active at 34 °C, but we recommend performing digests at room temperature (20 °C) or 4 °C. The activity of TEV protease is approximately 3-fold greater at 20 °C than at 4 °C [Nallamsetty et al., 2004]. It will tolerate a range of buffers, including phosphate, MES, and acetate. TEV protease is not adversely affected by the addition of glycerol or sorbitol (up to at least 40% w/v). TEV protease is sensitive to some detergents ([[http://www.sciencedirect.com/science/article/pii/S1046592802005892|Mohanty et al., 2003]]; [[http://www.sciencedirect.com/science/article/pii/S0003269708004909|Lundback et al., 2008]]).
-; Lundback et al., 2008].
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