Overview

Description

  • Nature
    Native
  • Source
    Native
  • Amino Acid Sequence
    • Species
      Human
    • Molecular weight
      30 kDa

Specifications

Our Abpromise guarantee covers the use of ab91099 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

  • Biological activity
    Activity: 20-22 units per mg protein. One unit is defined as the amount of enzyme that hydrolyzes one micromole of Meo-suc-ala-pro-val-pNA per minute at 25°C in 100 mM Tris-HCl, pH 7.5, with 500 mM NaCl.
  • Applications

    Western blot

    SDS-PAGE

    Functional Studies

  • Purity
    > 95 % SDS-PAGE.

  • Form
    Lyophilised
  • Additional notes
    Reconstitute in 50 mM Na acetate pH 5.5 with 150 mM NaCl. Dissolve at a concentration of 1 to 2 mg/mL and store at -20 degrees C. If not immediately frozen, this enzyme should be used promptly to limit loss of activity. We do not suggest repeated thawing/freezing cycles. Protein Determination: Extinction Coefficient (E) 0.1% at 280nm, 1cm pathway = 0.985. Prepared from whole blood shown to be non reactive for HBsAg, anti-HCV, anti- HBc, and negative for anti-HIV 1 & 2 by FDA approved tests.
  • Concentration information loading...

Preparation and Storage

  • Stability and Storage

    Shipped at 4°C. Store at -20ºC.

    Constituent: ddH20

    This product is an active protein and may elicit a biological response in vivo, handle with caution.

General Info

  • Alternative names
    • Bone marrow serine protease
    • ELA2
    • ELANE
    • Elastase 2
    • Elastase 2 neutrophil
    • Elastase neutrophil expressed
    • Elastase-2
    • ELNE_HUMAN
    • GE
    • Granulocyte derived elastase
    • HLE
    • HNE
    • Human leukocyte elastase
    • Leukocyte elastase
    • Medullasin
    • NE
    • Neutrophil elastase
    • PMN E
    • PMN elastase
    • Polymorphonuclear elastase
    • SCN1
    see all
  • Function
    Modifies the functions of natural killer cells, monocytes and granulocytes. Inhibits C5a-dependent neutrophil enzyme release and chemotaxis.
  • Tissue specificity
    Bone marrow cells.
  • Involvement in disease
    Defects in ELANE are a cause of cyclic haematopoiesis (CH) [MIM:162800]; also known as cyclic neutropenia. CH is an autosomal dominant disease in which blood-cell production from the bone marrow oscillates with 21-day periodicity. Circulating neutrophils vary between almost normal numbers and zero. During intervals of neutropenia, affected individuals are at risk for opportunistic infection. Monocytes, platelets, lymphocytes and reticulocytes also cycle with the same frequency.
    Defects in ELANE are the cause of neutropenia severe congenital autosomal dominant type 1 (SCN1) [MIM:202700]. SCN1 is a disorder of hematopoiesis characterized by a maturation arrest of granulopoiesis at the level of promyelocytes with peripheral blood absolute neutrophil counts below 0.5 x 10(9)/l and early onset of severe bacterial infections.
  • Sequence similarities
    Belongs to the peptidase S1 family. Elastase subfamily.
    Contains 1 peptidase S1 domain.
  • Information by UniProt

Images

  • SDS-PAGE: 12% Bis-Tris NuPAGE gel
    Lane 1: 5 μg Neutrophil Elastase (reduced/heated)
    Lane 2: 10 μg Neutrophil Elastase (reduced/heated)
    Lane 3: 20 μg Neutrophil Elastase (reduced/heated)
    Lane 4: Molecular weight markers

References

This product has been referenced in:
  • Wen G  et al. Genetic and Pharmacologic Inhibition of the Neutrophil Elastase Inhibits Experimental Atherosclerosis. J Am Heart Assoc 7:N/A (2018). Read more (PubMed: 29437605) »
  • Thorlacius-Ussing J  et al. Non-invasive profiling of protease-specific elastin turnover in lung cancer: biomarker potential. J Cancer Res Clin Oncol N/A:N/A (2018). Read more (PubMed: 30467633) »
See all 2 Publications for this product

Customer reviews and Q&As

1-7 of 7 Abreviews or Q&A

Question
Answer

We only have 3 active human neutrophilase elastase proteins in our catalogue:

https://www.abcam.com/active-human-neutrophil-elastase-full-length-protein-ab91099.html
https://www.abcam.com/active-human-neutrophil-elastase-full-length-protein-ab134433.html
https://www.abcam.com/active-human-neutrophil-elastase-full-length-protein-ab80475.html

I have been in contact with the labs to see whether any of these would be suitable for use in cell culture.

Ab91099: This product has NOT been tested for endotoxins. It is not cell culture grade and we do not recommend it for cell culture use.

ab80475: We cannot recommend Catalog # A50145H for use in cell culture as we do not test it for endotoxins nor is anything done to mitigate the presence of endotoxins in the course of its purification.

ab134433: Normally, due to biochemist/chromatography purification steps, our protein should not contain any endotoxins however, we didn't test this.

An infection of this protein could be easily checked by using Limulus Amebocyte Lysate (LAL-endotoxin) test. The easiest way is to make a plasma dilution 1:4 with 0,15mol/L NaCl and heat at 60 C for 30 min. This step is important to inactivate all inhibitors and give the possibility to perform the LAL test. Our enzyme is in 10 mM acetate buffer pH 5.5, it means that is good enough to be used for life cells/cell culture.

Out of these three products, I would therefore suggest you use ab134433 as it is the most suitable for your particular experiment.

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Question
Answer

Thank you for contacting us.

As a follow up from our phone call, the lab has confirmed that this protein has not been tested for endotoxins.


I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thats no problem. If you have any further questions, please do not hesitate to contact us again.

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Answer

I now have some further information to explain the 14 kDa band observed in both the SDS page gel and Western blot of Neutrophil Elastase protein (Active), ab91099.

I have been informed by the lab that performing SDS-page under reducing conditions with Human Neutrophil Elastase (HNE) is difficult. The reason for this is that the sample buffer is of a neutral pH. This is the optimal pH for HNE activity. Pure HNE will undergo autolysis when the substrate is not present. The 14 kDa is the resulting autolytic HNE peptide and is detected by the anti-HNE antibody used in Western blotting. This is consistent with more of this product being present at higher concentrations of protein. In order to prevent this degradation, an HNE inhibitor can be added prior to running the SDS-page or performing native gel electrophoresis at an acidic pH.

I hope this information has reassured you over the quality of the product. If I can help in any further way please do let me know.

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Answer

I understand your concerns in regards to this second band. From the SDS-page gel, it appears that the band at ˜14 kDa also increases with increasing concentration (it is quite low in the 5 ug lane). It therefore appears that the band is concentration dependent which suggests that it is not a contamination with a different protein, but an entity with is formed when the concentration is increase.
I cannot explain why this is happening and I am waiting on further information from the lab in exactly what it is. I will let you know as soon as I have this information to share with you. I am sorry for the delay.

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Answer

Thank you for providing me with that extra information.
I now have some more information to share with you. It seems that the unexpected band at 14 kDa is more likely to be observed in the western blot if the sample is overloaded with protein. As can be seen from the western blot image I have attached to this email (produced from the same western blot presented on the datasheet of ab91099), using 0.1 ug of the protein (lane 1), compared to 0.01 ug (lane 2) does not significantly increase the expected signal at ˜25 kDa, but the band at ˜14 kDa is significantly increased in intensity. This increase in the 14 kDa band with protein concentration can also be seen in the SDS page gel presented on the datasheet.
I would therefore suggest that you reduce the amount of protein loaded on the gel (trying 0.01 and 0.1 ug initially).
The western blot presented on the datasheet of ab91099 was performed as follows:
The membrane is blocked for 1 hour at room temperature with 5% BSA in 0.1% TBST, with gentle rocking. The primary was with diluted with 5% BSA in 0.1% TBST and incubated overnight at 4oC with gentle rocking.
I am still waiting on further information as to why the 14 kDa band is formed. I will get back to you with this information as soon as I can.
I hope this information has been of help, if you have any questions please do not hesitate to ask.

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Answer

Thank you for providing that information. I now understand more fully what your concerns regarding the protein are.
It seems that the lower band is much more prominent (in comparison to the expected ˜25 kDa band) when blocked with BSA and a higher concentration of the antibody is used. I am still waiting for further information about what the lower band ˜14 kDa is.
I have a few further questions if you wouldn't mind:
1. What antibody have you been using to detect the Neutrophil Elastase protein?
2. How were the samples prepared prior to loading on the gel? What loading buffer was used (reducing conditions used or not) and were the samples boiled?
I will let you know as soon as I have some further information to share with you.

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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