Overview

  • Product name
    Anti-TNPO3 antibody [3152C2a]
    See all TNPO3 primary antibodies
  • Description
    Mouse monoclonal [3152C2a] to TNPO3
  • Host species
    Mouse
  • Tested applications
    Suitable for: Flow Cyt, WB, Dot blot, IHC-P, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Recombinant fragment (Human) from near the N terminus.

  • Positive control
    • HeLa whole cell lysate (ab150035); NIH3T3 whole cell lysate

Properties

Applications

Our Abpromise guarantee covers the use of ab54353 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
Flow Cyt Use 1µg for 106 cells.

ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.

 

WB 1/50 - 1/100. Detects a band of approximately 90 kDa (predicted molecular weight: 110 kDa).
Dot blot Use at an assay dependent concentration.
IHC-P Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF 1/300.

Target

Images

  • Anti-TNPO3 antibody [3152C2a] (ab54353) at 1/100 dilution + HeLa whole cell lysate at 25 µg

    Secondary
    anti Mouse IgG at 1/2500 dilution

    Predicted band size: 110 kDa
    Observed band size: ~90 kDa
    why is the actual band size different from the predicted?

  • Anti-TNPO3 antibody [3152C2a] (ab54353) at 1/50 dilution + NIH3T3 whole cell lysate at 25 µg

    Secondary
    anti Mouse IgG at 1/2500 dilution

    Predicted band size: 110 kDa
    Observed band size: ~90 kDa why is the actual band size different from the predicted?

  • IHC image of ab54353 staining in human normal cervix formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab54353, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

  • Overlay histogram showing HeLa cells stained with ab54353 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab54353, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 80% methanol/permeabilized in 0.1% PBS-Tween used under the same conditions.

References

This product has been referenced in:
  • Jovicic A  et al. Modifiers of C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS. Nat Neurosci 18:1226-9 (2015). Read more (PubMed: 26308983) »
  • Konstantoulas CJ & Indik S Mouse mammary tumor virus-based vector transduces non-dividing cells, enters the nucleus via a TNPO3-independent pathway and integrates in a less biased fashion than other retroviruses. Retrovirology 11:34 (2014). WB . Read more (PubMed: 24779422) »
See all 16 Publications for this product

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

Read More

Question

Thank you for the reply.

Here are the answers for your question.

- You mention you are using muscle tissue - from what species? Is the tissue paraffin embedded? How thick are the sections?

I used frozen both human muscle and paraffin-embedded formalin-fixed mouse muscle.

The specimens were made at 6 micrometer thick.

The conditions of fixation I tested are as below.

1. Frozen human muscle, unfixed

2. Frozen human muscle, formalin-fixed, with and without heat-induced antigen retrieval

3. Frozen human muscle, acetone fixed (-20 degrees Celsius), with and without heat-induced antigen retrieval

4. Paraffin-embedded formalin-fixed mouse muscle, with heat-induced antigen retrieval



- What methods of antigen retrieval (buffer, heat, enzyme)have you tested? What blocking reagents were used? Time and temperature of incubation?Was the primary antibody diluted in block? Time and temperature of incubation?

I used heat-induced antigen retrieval.

The specimens were incubated within boiled citrate buffer (pH 6.0) for 15 minutes.

Then they were blocked with 1% BSA at room temperature for 30 minutes.

The primary antibody was diluted in 1% BSA, and the specimens were incubated with the primary antibody at room temperature for 1 hour, or at 4 degrees Celsius for overnight.


- Finally, what is the secondary antibody being used? Do you know that it works with other primary antibodies?

I used biotinylated anti-mouse IgG antibody as the secondary antibody.

I tested with anti-dystrophin antibody prior to the experiment with anti-TNPO-3 antibody, and it worked.


I am looking forward your advice. Thank you.

Read More
Answer

Thank you for your reply with the requested information.


Based on the details you provided, I believe this antibody is not working as we would expect. Although the product has not been specifically validated for frozen sections, I would expect signal from paraffin embedded mouse tissues using antigen retrieval. The only suggestion I can make is to add tween or triton to your buffers if they are not already present in order to help better permeabilize the tissue.

Can you please provide an order number for this antibody? If you are contacting us within 6 months of purchase, I am happy to offer a replacement from another lot or a credit. Please let me know which you would prefer.


I look forward to your reply so that I may assist you further. Please do not hesitate to contact me if you have any additional questions or concerns.

Read More

Answer

Thank you for contacting Abcam regarding ab54353.


I am sorry that you have been experiencing difficulties with this antibody in IHC. In order to assist you further, I was hoping you would be able to provide some additional information regarding the samples you tested and the protocol you used.


You mention you are using muscle tissue - from what species? Is the tissue paraffin embedded? How think are the sections? What methods of antigen retrieval (buffer, heat, enzyme) have you tested?


What blocking reagents were used? Time and temperature of incubation? Was the primary antibody diluted in block? Time and temperature of incubation?


Finally, what is the secondary antibody being used? Do you know that it works with other primary antibodies?

I look forward to your reply so that I may assist you further. Please do not hesitate to contact me if you have any additional questions.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (ES cells)
Loading amount
100000 cells
Specification
ES cells
Gel Running Conditions
Non-reduced Denaturing (NuPage 4-12% Bis-Tris)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Apr 23 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Hela cells)
Specification
Hela cells
Fixative
Methanol
Permeabilization
No
Blocking step
Fish scale gelatin in PBS as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 0.2% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Aug 11 2010

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

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