Overview

  • Product name
  • Description
    Rabbit polyclonal to DNase II
  • Host species
    Rabbit
  • Specificity
    Specific for the 32 kD alpha chain; does not recognise the 12 kD beta chain.
  • Tested applications
    Suitable for: ICC/IF, IHC-P, WBmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Peptide corresponding to amino acids 347 to 360 of human DNase II precursor.

  • Positive control
    • Human spleen tissue lysate
  • General notes


    Apoptosis is characterized by several morphological nuclear changes including chromatin condensation and nuclear fragmentation. These changes are triggered by the activation of members of caspase family, caspase activated DNase, and several novel proteins including AIF and Acinus. DNase II causes both chromatin condensation and DNAfragmentation (Zamzami & Kroemer, 1999). The genes encoding human (Yasuda et al.1998, Shiokawa & Tanuma, 1998, Baker et al, 1998), porcine, and murine DNase II have been cloned (Krieser & Eastman, 1998). The DNaseII gene encodes a 40 kDa proenzyme. The mature enzymeconsists of two non-identical subunits, the 32 kDa (a) and 12kDa (b) chains, generated by proteolytic processing. Overexpression of DNase II induces chromatin condensation (Shiokawa & Tanuma, 1998). DNase II is ubiquitously expressed in human tissues.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
  • Storage buffer
    Preservative: 0.02% Sodium azide
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Primary antibody notes
    Apoptosis is characterized by several morphological nuclear changes including chromatin condensation and nuclear fragmentation. These changes are triggered by the activation of members of caspase family, caspase activated DNase, and several novel proteins including AIF and Acinus. DNase II causes both chromatin condensation and DNAfragmentation (Zamzami & Kroemer, 1999). The genes encoding human (Yasuda et al.1998, Shiokawa & Tanuma, 1998, Baker et al, 1998), porcine, and murine DNase II have been cloned (Krieser & Eastman, 1998). The DNaseII gene encodes a 40 kDa proenzyme. The mature enzymeconsists of two non-identical subunits, the 32 kDa (a) and 12kDa (b) chains, generated by proteolytic processing. Overexpression of DNase II induces chromatin condensation (Shiokawa & Tanuma, 1998). DNase II is ubiquitously expressed in human tissues.
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab8119 in the following tested applications.

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

Application Abreviews Notes
ICC/IF Use a concentration of 5 µg/ml.
IHC-P Use a concentration of 5 µg/ml.
WB 1/500 - 1/1000. Detects a band of approximately 32 kDa (predicted molecular weight: 36 (precursor) kDa).Can be blocked with DNase II peptide (ab8373).

Target

  • Function
    Hydrolyzes DNA under acidic conditions with a preference for double-stranded DNA. Plays a major role in the degradation of nuclear DNA in cellular apoptosis during development. Necessary for proper fetal development and for definitive erythropoiesis in fetal liver, where it degrades nuclear DNA expelled from erythroid precursor cells.
  • Sequence similarities
    Belongs to the DNase II family.
  • Post-translational
    modifications
    Glycosylated. Mutations that eliminate N-glycosylation sites reduce activity, but enzymatic deglycosylation has no effect.
  • Cellular localization
    Lysosome.
  • Information by UniProt
  • Database links
  • Alternative names
    • Acid DNase antibody
    • Deoxyribonuclease 2 alpha antibody
    • Deoxyribonuclease 2 antibody
    • Deoxyribonuclease II alpha antibody
    • Deoxyribonuclease II antibody
    • Deoxyribonuclease II lysosomal antibody
    • Deoxyribonuclease-2-alpha antibody
    • Deoxyribonuclease2 antibody
    • DeoxyribonucleaseII antibody
    • DNASE 2 antibody
    • DNASE 2A antibody
    • DNase II alpha antibody
    • DNase II lysosomal antibody
    • DNASE2 antibody
    • DNASE2A antibody
    • DNaseII antibody
    • DNL 2 antibody
    • DNL antibody
    • DNL2 antibody
    • DNS2A_HUMAN antibody
    • Lysosomal DNase II antibody
    • R31240 2 antibody
    • R31240_2 antibody
    see all

Images

  • Immunohistochemistry (Formalin-fixed paraffin embedded sections) of human spleen tissue labeling DNase II with Anti-DNase II antibody (ab8119) at 5μg/ml.
  • All lanes : Anti-DNase II antibody (ab8119) at 1/500 dilution

    Lane 1 : Human spleen tissue lysate with absence of blocking peptide
    Lane 2 : Human spleen tissue lysate with presence of blocking peptide

    Predicted band size: 36 (precursor) kDa
    Observed band size: 40 kDa
    why is the actual band size different from the predicted?

  • ab8119 at 5µg/ml staining DNase II in human spleen tissue by IHC

  • ICC/IF image of ab8119 stained Hek293 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab8119, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

References

This product has been referenced in:
See all 2 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Answer

Thank you for contacting us and your interest in our products.

As discussed over the phone, the anti-DNase II antibody (ab8119) has to our knowledge not been used with mouse samples. If we have conclusive information to show that a particular antibodyis not suitable for aspecies or application, we would state this on the datasheet.

However, having aligned the mouse and human DNase II protein sequences (SwissProt references http://www.uniprot.org/uniprot/P56542and http://www.uniprot.org/uniprot/O00115respectively), I would not expect the antibody to detect the mouse protein. The immunogen used is the C-terminal residues 347-360 of the human protein (highlighted in yellow in the attachment of this email). Unfortunately, the mouse protein significantly diverges from the human protein in this region.

Unfortunately, the other antibody we have against this target is also raised against this region and would therefore also not be predicted to work. I am sorry that I could not be of more help in this instance. You may have more luck if you find an antibody raised against the N-terminal region of the protein. You may find the following website useful in this search:

http://www.biocompare.com

I hope this information has been of some help. If you require any further information please do not hesitate to contact us again.

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Question

LOT NUMBER -- NOT SPECIFIED --
ORDER NUMBER -- NOT SPECIFIED --
DESCRIPTION OF THE PROBLEM Multiple bands, also purchased spleen lysate (ab29699) recommended by abcam, however there are no obvious bands almost a smear on the gel, when adjusting the levels in photoshop can see bands. My own cell lysate has numerous bands however by far the strongest signal comes from a band at about 60 kDa which is bigger than expected.
SAMPLE Primary human fibroblast cell culture
PRIMARY ANTIBODY Abcam, rabbit polyclonal, PBS with 0.02% sodium azide, 1:500, overnight at 4 C, washed 3 x times quickly followed by 3 x times 15 minutes in TBST (0.1% tween)at room temperature.
DETECTION METHOD ECL plex
POSITIVE AND NEGATIVE CONTROLS USED Positive - spleen lysate ab29699 nuclear extract expected to be negative (lanes run with loading buffer showed no bands)
ANTIBODY STORAGE CONDITIONS aliquoted into 10ul samples and stored at -20 C.
SAMPLE PREPARATION protein extracted using active motif kit protease and phosphatase inhibitors present, sample heated to 95 C in loading buffer (containing 2-mercaptoethanol)
AMOUNT OF PROTEIN LOADED 30 ug
ELECTROPHORESIS/GEL CONDITIONS reducing gel, 4 % stacking gel 12% seperating gel acrylamide
TRANSFER AND BLOCKING CONDITIONS transfer buffer (glycine, tris base, 20% methanol), blocking 5% non-fat milk in TBST (0.1% tween).
SECONDARY ANTIBODY GE healthcare, goat anti rabbit, ddH2O (1ug/ul), 1:2500, 1 hr at RT, washed 3 x TBST (0.1% tween) quick followed by 3 x TBST 15 minutes.
HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2
HAVE YOU RUN A "NO PRIMARY" CONTROL? No
DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes
WHAT STEPS HAVE YOU ALTERED? different primary dilution
ADDITIONAL NOTES In the attached document: WCE = whole cell extract, CYTO = cytoplasmic extract, NUC = nuclear extract, M = marker.

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Answer

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

I would like to reassure you that ab8119 is tested and covered by our 6 month guarantee for use in western blot and human samples. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement. The lysate ab29699 is also covered by the guarantee.

Reviewing this case, I would like to offer some suggestions to help optimise the results. I would also appreciate if you can confirm some further details which will help me in my investigations:

1. I am sorry to confirm regrettably the attached file did not come through. I would appreciate if you are able to resend this?

2. I notice your research institute have purchased this twice, could you confirm if the first vial worked, and if this was on the same sample type?

3. I can suggest to try the antibody at a lower concentration, 1:1000, to reduce the background and help provide more consistent results between batches.

4. Could you confirm if the secondary antibody is working well with other primary antibodies. I can recommend it would be beneficial to consider including a no primary control to assess if the secondary is binding non specifically. The concentration of the secondary may need to be reduced in order to optimize the results.

5. Regarding the ab29699 Spleen lysate, could you confirm if this has been run on an SDS gel and stained with coomassie to assess the quality? I would appreciate if you are able to provide an image.

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

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Answer

Thank you for contacting Abcam. These antibodies are almost identical and so either would suit your needs. The only difference I can see is that ab8119 does not recognize the 12 kD beta chain, while it is not specified for ab115233. Currently ab115233 is out of stock and is due back in stock in 1-2 weeks, whereas ab8119 is available immediately. For a positive control in western blot, I would recommend human spleen tissue, ab29699 or human cerebellum tissue, ab30062. If there is anything else I can help you with, please let me know.

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