Overview

  • Product name
    Anti-O-Linked N-Acetylglucosamine antibody [RL2]
    See all O-Linked N-Acetylglucosamine primary antibodies
  • Description
    Mouse monoclonal [RL2] to O-Linked N-Acetylglucosamine
  • Host species
    Mouse
  • Specificity
    Detects nuclear pore complex (NPC), cytoplasmic and intranuclear O-linked glycoproteins from human, mouse, and rat tissues. In Western blot many bands are expected as the O-Linked N-Acetylglucosamine modification can occur on proteins of different sizes.
  • Tested applications
    Suitable for: ICC/IF, IHC-Fr, ChIP/Chip, Dot blot, WB, IPmore details
  • Immunogen

    Pore complex-lamina fraction purified from rat liver nuclear envelopes.

  • Positive control
    • ICC-IF: MCF7 cells. WB: Jurkat cells treated with 50 uM PugNAc; SH-SY5Y) whole cell lysate - treated with 50µM z-Pugnac; Rat Liver Nuclear Envelope lysate.
  • General notes

    This antibody clone [RL2] is manufactured by Abcam.

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

Properties

Applications

Our Abpromise guarantee covers the use of ab2739 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 - 10 µg/ml.
IHC-Fr Use at an assay dependent concentration. PubMed: 23734074
ChIP/Chip Use at an assay dependent concentration. PubMed: 20368426
Dot blot 1/800.
WB Use a concentration of 1 µg/ml.
IP Use at an assay dependent concentration.

Target

  • Relevance
    Many cellular proteins, including nuclear pore, oncogene, cytoskeletal, heat shock, viral and transcription regulatory proteins contain single O-linked N-acetylglucosamine (O-GlcNAc) residues attached to serine or threonine residues. It has been observed that O-GlcNAc glycosylated proteins tend to be under phosphorylated relative to unglycosylated proteins and that O-GlcNAc bearing proteins tend to be found in multimeric complexes. This has led to the suggestion that O-GlcNAc glycosylation may obscure phosphorylation sites and acts as a signaling mechanism or mediator of signaling.

Images

  • All lanes : Anti-O-Linked N-Acetylglucosamine antibody [RL2] (ab2739) at 1 µg/ml

    Lanes 1 & 3 : Jurkat cells treated with 0 uM PugNAc
    Lane 2 : Jurkat cells treated with 50 uM PugNAc (3 hours)
    Lane 4 : Jurkat cells treated with 4 mM glucosamine and 50 uM PugNAc (3 hours)

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/50000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Exposure time: 12 minutes


    Jurkat cells were treated with either 50 uM PugNAc (ab144670) or 4 mM glucosamine + 50 uM PugNAc (ab144670) for three hours prior to harvest to stimulate O-linked glycosylation. The expected increase in glycosylation is observed in the treated lanes 2 & 4. 

  • ab2739 stained in MCF7 cells. Cells were fixed with 4% paraformaldehyde (10min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab2739 at 5µg/ml and ab6046 (Rabbit polyclonal to beta Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150080 (pseudo-colored red) and ab150117 (colored green) used at 1 ug/ml for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature.

  • All lanes : Anti-O-Linked N-Acetylglucosamine antibody [RL2] (ab2739) at 1/3000 dilution

    Lane 1 : Human neuroblastoma (SH-SY5Y) whole cell lysate - treated with 50µM z-Pugnac for 24 hours
    Lane 2 : Human neuroblastoma (SH-SY5Y) whole cell lysate - untreated

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : HRP-conjugated horse anti-mouse IgG polyclonal

    Developed using the ECL technique.

    Performed under reducing conditions.

    Exposure time: 30 seconds

    See Abreview

  • Anti-O-Linked N-Acetylglucosamine antibody [RL2] (ab2739) at 1 µg/ml + Rat Liver Nuclear Envelope at 10 µg

    Secondary
    Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Exposure time: 1 minute


    The antibody was tested against the immunogen (isolated rat liver nuclear envelopes, which contain 8 O-linked glycoproteins in the nuclear pore complex).

Protocols

References

This product has been referenced in:
  • Brown HM  et al. Periconception onset diabetes is associated with embryopathy and fetal growth retardation, reproductive tract hyperglycosylation and impaired immune adaptation to pregnancy. Sci Rep 8:2114 (2018). IHC ; Mouse . Read more (PubMed: 29391475) »
  • Shi H  et al. Skeletal muscle O-GlcNAc transferase is important for muscle energy homeostasis and whole-body insulin sensitivity. Mol Metab N/A:N/A (2018). Read more (PubMed: 29525407) »
See all 66 Publications for this product

Customer reviews and Q&As

1-10 of 10 Q&A

Answer

You are correct that the uniprot link in the previous email (Uniprot: http://www.uniprot.org/uniprot/Q6ZMB0http://www.uniprot.org/uniprot/Q6ZMB0) is for the core 3 synthase, which is B3GNT6. Inferring from the alternative names for this synthase (Beta-1,3-N-acetylglucosaminyltransferase 6) is how the relation to ab2735 was established, since ab2735 recognizes beta-1,3 linked O-linked N-acetylglucosamine (O-GlcNAc).

However, this information does not guarantee that an anti O-GlcNAc antibody such as ab2735 will be specific for core 3 O-glycan, as core 2 and core 4 are also formed by the addition of N-acetyl-glucosamine.

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Answer

I can confirm that the B3GNT6 antibodies, https://www.abcam.com/b3gnt6-antibody-ab103239.html& https://www.abcam.com/b3gnt6-antibody-ab168600.html, will recognize the transferase that synthesizes the core 3 structure of the O-glycan. We also currently offer 2 antibodies, https://www.abcam.com/O-Linked-N-Acetylglucosamine-antibody-HGAC85-ab2735.html and https://www.abcam.com/o-linked-n-acetylglucosamine-antibody-rl2-chip-grade-ab2739.html, against Anti-O-Linked N-Acetylglucosamine. https://www.abcam.com/O-Linked-N-Acetylglucosamine-antibody-HGAC85-ab2735.html shows specificity for the Core 3 synthase (Uniprot: http://www.uniprot.org/uniprot/Q6ZMB0http://www.uniprot.org/uniprot/Q6ZMB0).

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Answer

Thank you for contacting us about this issue. Have you successfully stained other nuclear proteins using this protocol and these reagents? I think the protocol looks correct, but I am concerned the permeabilization of the nuclei may not be effective. We have not had reports of instability but our recommendation is to store the antibody long-term at -20C or -80C. It is possible that there is still enough activity for the antibody to work in western blotting but not in ICC/IF.

Is the secondary antibody known to be good?

I look forward to your reply.

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Answer

Thank you for your swift reply. With regard of the new information I would like to suggest the following alterations in order to increase the signal strength: 1. We found that many monoclonal antibodies work better with BSA than milk as a blocking agent (see attachment). I would therefore suggest to switch to 3%BSA in TBS/TBST during blocking/incubation. 2. In order to prevent a washing off bound antibodies, I would like to suggest decreasing the washing time down to 3x 5 min after the primary antibody. Should the suggestions not improve the results, please do let me know. In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement or credit note. I hope this information is helpful, and I thank you for your cooperation.

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Question

Hello ,



Here is a complain from customer for no bands







3) Description of the problem (high background, wrong band size, more bands, no band etc):
Sensitivity of the antibody too low. Very faint or no bands

4) Antibody storage conditions (temperature/reconstitution etc):

Aliquots at -20
5) a)Sample (Species):

Mouse and human cell lines


b) Type of sample (Cell extract/Nuclear extract/Purified protein/Recombinant protein etc).:

Immunoprecipitated protein and cell lysate

6) a) Sample preparation:
Lysis buffer
laemmli
Protease inhibitors
yes
Phosphatase inhibitors
yes
Reducing agent
yes
Boiling for ≥5 min?

yes

b) Amount of protein loaded:


Protein loaded ug/lane or cells/lane
50-100 microgms

7) a) Electrophoresis/Gel conditions:
Reducing or Non Reducing gel

reducing

Percentage of gel

8-10
Volts applied
75

Time applied
4 hrs


b) Transfer or blocking conditions:

Type of membrane
PVDF

Protein transfer verified
yes

Blocking agent and concentration
Milk 5%


Blocking time

1 hr

Blocking temperature
RT


8) Primary antibody Concentration or dilution

1:500
Diluent buffer
Tris buffered saline

Incubation time
1 hr or 4 degree centi

Incubation temperature
1 Hr or O/N


Washing: Buffer Used
Tris buffered saline
Number of washes
3 washes

9) Secondary antibody:

Species
rabbit

Reacts against
mouse

Concentration or dilution
1:2000

Diluent buffer
Tris buffered saline
Incubation time
1 hr

Incubation temperature:

RT


Fluorochrome or enzyme conjugate
Enyme HRP


Washing: Buffer Used
Tris buffered saline

Number of washes
3

10) Detection method (ECL, ECL plus, etc.):
Roche chemiluminiscence

11) Positive and negative controls used

Positive control
Nuclear lysate

Negative control
IgG control

12) a) How many times you have run this staining?

7 to 8 times


b) Do you obtain the same results every time?


yes

C) What steps have you altered to try and optimize the use of this antibody?
Varying dilution, washes, incubation time, amount of protein, detection reagent

Read More
Answer

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.
The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.
Having reviewed this case, I would appreciate if you can confirm some further details to help optimize the results from ab2739 Anti-O-Linked N-Acetylglucosamine antibody [RL2] - ChIP Grade:
1. Do you boil your samples? If so, how long and at which temperature?
2. Are you checking the transfer with Ponceau?
3.What kind of blocking agent are you using during antibody incubation and at what concentration?
4. How long do you wash the blot after incubation?
Should the suggestions not improve the results, please do let me know.
In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.
I hope this information is helpful, and I thank you for your cooperation.

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Question
Answer

Thanks for your inquiry. The clone number for ab2739 is RL2. It's highly probable that ab2739 would work in IHC as it has been successfully tested in ChIP,ICC/IF, IP, and WB. If you are interested, you could submit an abreview of ab2739 using the untested IHC. Below is more information on how to do it: Thank you very much for your interest in ab2739. To our knowledge, ab2739 has not been tested in IHC. Therefore, I can offer a discount off a future purchase if you buy ab2739 now, test it in IHC and submit feedback to us in the form of an Abreview. It doesn’t matter whether the Abreview is positive or negative, we would just really like to receive your feedback. The discount would be to the value of 1 free primary antibody. If you are interested in this offer, please follow these steps: 1. Reply to this e-mail to let me know that you would like to proceed and test ab2739 in IHC. I will then send a discount code. This code must be issued before purchasing ab2739 so please wait for my reply before ordering. 2. Purchase ab2739 either by phone, fax, or online (www.abcam.com). 3. Test it in IHC. 4. Let us know the results, positive or negative, using our Abreview system (this will take about 10 minutes and images are great if you have them!). To find out more about our Abreview system, please visit: https://www.abcam.com/abreviews. 5. After the review is submitted to us, the discount code becomes active. Simply place your new order by phone, fax, or on the web and mention the discount code. The discount can be redeemed for any primary antibody ordered and the discount code is valid for 4 months after issue. We are always pleased to obtain feedback about our products and any information is greatly appreciated! Even if ab2739 turns out to be unsuitable for IHC, you will still receive the discount on your next purchase after your Abreview has been submitted. Please let me know if you have any questions about this offer and I would be happy to help you further. The Terms and Conditions of this offer can be found at: www.abcam.com/collaborationdiscount.

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Answer

Thank you for contacting us. You may find the following reference useful: Mapping Sites of O-GlcNAc Modification Using Affinity Tags for Serine and Threonine Post-translational Modifications* Lance Wells et al, Molecular & Cellular Proteomics 1.10 791 They use 10uM PUGNAc (O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate) added with the protease inhibitors during sample preparation. I hope this helps, please let me know if you need any additional information.

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Answer

Thank you for contacting us. The following reference from 1987 describes the production and characterization of the RL2 clone, among others: Snow CM et al. Monoclonal antibodies identify a group of nuclear pore complex glycoproteins. J Cell Biol 104:1143-56 (1987). PubMed: 2437126 http://jcb.rupress.org/content/104/5/1143.long The paper does not report, however, specificity for either serine or threonine O-linkages. Another paper from this group describes more characterization of RL2: Holt GD et al. Nuclear pore complex glycoproteins contain cytoplasmically disposed O-linked N-acetylglucosamine. J Cell Biol 104:1157-64 (1987). PubMed: 3571327 http://jcb.rupress.org/content/104/5/1157.full.pdf but there is again no mention of specificity, only a note in the Discussion section (page 1163) stating that "The precise composition of these epitopes... will be determined in future studies". 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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Question
Answer

Thank you for your enquiry. The concentration of this lot is 2.0 mg/ml. Please contact us again if you have any additional questions.

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Question
Answer

The concentration is 2.0 mg/ml.

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