{"product_id":"neb-p8104s","title":"New England Biolabs, P8104S, Endoproteinase AspN","description":"Endoproteinase AspN (flavastacin) is a zinc metalloendopeptidase which selectively cleaves protein and peptide bonds N-terminal to aspartic acid residues.  \u003cb\u003eRelated Categories\u003c\/b\u003e Proteases,, Proteome Analysis  \u003cb\u003eApplications\u003c\/b\u003e Glycomics and glycoproteomics,, Biotherapeutics and Antibody Analysis,, Proteomics  \u003cb\u003eSpecification\u003c\/b\u003e \u003cb\u003eReaction Conditions\u003c\/b\u003e 1X Endoproteinase AspN Reaction Buffer Incubate at 37°C 1X Endoproteinase AspN Reaction Buffer 50 mM Tris-HCl 2.5 mM ZnSO4 (pH 8 @ 25°C)  \u003cb\u003eUsage Concentration\u003c\/b\u003e 100 ng\/μl  \u003cb\u003eMolecular Weight\u003c\/b\u003e Theoretical: 40089.9 daltons  \u003cb\u003eFAQ\u003c\/b\u003e Q: Is your Endoproteinase AspN the same as the Endoproteinase AspN sold by other companies? A: No, it is different from other Endoproteinase AspN's available on the market. They are from different strain sources, so they’re different enzymes with similar specificities. NEB's Endoproteinase AspN digests proteins and peptides rapidly, but digests peptides more rapidly. NEB's Endoproteinase AspN was first characterized in 1994 by Tarentino. et. al. and is found in the MEROPS database. Q: The Endoproteinase AspN is not fully dissolving when I reconstitute it in water. How do I get it completely into solution? A: There is sometimes a precipitate observed in this product, but it does not seem to affect the activity. We recommend mixing and keeping the tube on ice for 10 minutes, followed by a quick spin and then using the enzyme even if the precipitate is still present. Q: Which residues does Endoproteinase AspN cut? A: We have only observed Endoproteinase AspN cleaving at Asp residues. Q: Do you have a protocol or pointers for digesting proteins in-gel with endo- proteinase Asp-N followed by Endoproteinase Glu-C? I'm following the protocol for each but would like to streamline the combined digest. A: We suggest mixing the two enzyme buffers 1:1 GluC:AspN buffer. The Endo- proteinase GluC would prefer to have the E-E dipeptide present to stimulate activity and Endoproteinase AspN requires Zn, which is present in the AspN buffer. Still use a 1:20 (wt\/wt) ratio of enzyme to substrate for each enzyme. There will be some reduction in the cleavage of your substrate protein since the two proteases will be chewing each other. It has been our experience that these conditions work well enough to get a high degree of digestion. You may need to adjust the individual amounts of each enzyme if you are missing a desired fragment, assuming that the reactions will be analyzed by some MS technique. If you prefer to do the reactions sequentially, do the Endoproteinase GluC reaction first as Endoproteinase AspN has difficulty digesting intact pro- teins. All digestions should be performed at 37°C . Q: How pure is Endoproteinase AspN? Has it been checked for nuclease activity? A: The enzyme is provided in a nearly homogeneous form and may be free of nucleases but we have no data to support this. We presently have no QC test in place to qualify whether the Endoproteinase AspN is free of nuclease contamination. This product is intended to be used in Proteomic applications where generally nucleases are not an issue. Q: Tris will interfere with my reactions downstream. Does your Endoproteinase AspN, when reconstituted in H2O, contain Tris-HCl? A: The answer is yes. Prior to dry down the Endoproteinase AspN is dialyzed into 50mM NaCl, 10mM Tris-HCl buffer. Q: What is the Endoproteinase AspN sequence in text format? A: Here is the sequence of Endoproteinase AspN: TIVSSFIKTWPNATVYYTLPSQGSLSTQAYNTFLTNINKAFDMISSKTSV KFVQRTNQTEYITFTYSTGNSSPLGWVKNRVNGIKIYNTTYPAIIAHEIM HSMGIMHEQCRPDRDQYIIVDTNRAQDGTRHNFNLYNDYAGHGEFDFGSV MMYKSTDFAIDPNLPVMTKLDGSTFGKQRDGLSAGDYAGINHLYGPVNST SATNGTYTLTTSLAGDKNIDITGSSTADGTDVILYSATTGNNQKFIFRKS EHGYFTIKSILDSTKVLTVRNNGTANGTAVELRTNADTDAQKWLLFNLGN EGFGFAPKNAPSLRLEVKDGLTTNLTPIVIGSTDQTLQPYTKQRFTLTKVN Q: Can Endoproteinase AspN be used with a volatile buffer? A: Probably yes, but the reaction must be supplemented with zinc. Q: How stable is this enzyme? We dissolved our enzyme in water approx 2 weeks ago and the literature indicates that activity will start to drop after this length of storage time(and longer). A: Endoproteinase AspN stored at -20°C that has been freeze-thawed more than a dozen times over a two week period should have good activity. The Endoproteinase AspN enzyme we sell is an apo-enzyme and lacks the zinc required for activity. We add zinc to our buffer to reactivate the enzyme (hence the good shelf life). Q: I digested BSA with Endoproteinase AspN following standard conditions and examined the released fragments with Mass-spec. I did not see many peaks that matched the predicted fragment but saw a lot of unpredicted bands, perhaps indicating non-specific enzyme activity. A: This is more likely a case of poor or no digestion than non-specific digestion as our Endoproteinase AspN works best with peptides. Alternatively, you can do a \"double digest\": digest your protein first with Trypsin or Endoproteinase GluC to get it down to a smaller size, followed by or in combination with an Endoproteinase AspN digestion to generate peptides with N-terminal Aspartic acid. Q: Can endoproteinase AspN be heat inactivated? A: Endoproteinase AspN is a metalloenzyme which uses Zn+2 ions, therefore, we recommend inactivating it with the addition of EDTA at a greater concentration than the divalent cations that are present in the reaction buffer. This reaction is reversible if you add more Zn+2 or other divalent cations that will titrate out the EDTA. Q: I can’t get Endoproteinase AspN to digest my protein. A: Our Endoproteinase Asp N works better on peptide substrates than on intact proteins, so we suggest either using another enzyme to first generate large peptides that Endoproteinase AspN will have less trouble digesting (a double digest with either Trypsin or Endoproteinase GluC) or switching to another source of Endoproteinase AspN as our Endoproteinase AspN enzyme is different from other commercial sources. ","brand":"New England Biolabs","offers":[{"title":"Default Title","offer_id":46835492257961,"sku":"P8104S","price":0.99,"currency_code":"USD","in_stock":true}],"url":"https:\/\/iright.com\/products\/neb-p8104s","provider":"Iright","version":"1.0","type":"link"}