Overview

  • Product name
  • Description
    Rabbit polyclonal to TBX5
  • Host species
    Rabbit
  • Specificity
    This antibody is specific for TBX5.
  • Tested applications
    Suitable for: IHC-P, WBmore details
  • Species reactivity
    Reacts with: Human
    Predicted to work with: Mouse, Rat, Sheep, Rabbit, Goat, Horse, Chicken, Guinea pig, Cow, Cat, Dog, Zebrafish
  • Immunogen

    Synthetic peptide found within the following sequence: NSMHKYQPRL HIVKADENNG FGSKNTAFCT HVFPETAFIA VTSYQNHKIT (Human)

  • Positive control
    • HepG2 cell lysate.

Properties

Applications

Our Abpromise guarantee covers the use of ab49308 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
IHC-P Use a concentration of 5 µg/ml.
WB Use a concentration of 5 µg/ml. Detects a band of approximately 50 kDa (predicted molecular weight: 53 kDa). Good results were obtained when blocked with 5% non-fat dry milk in 0.05% PBS-T.

Target

Images

  • Lane 1 : Marker
    Lane 2 : Anti-TBX5 antibody (ab49308) at 5 µg/ml

    Lane 2 : HepG2 cell lysate at 10 µg/ml

    Secondary
    Lane 2 : HRP conjugated anti-Rabbit IgG at 1/50000 dilution

    Predicted band size: 53 kDa
    Observed band size: 50 kDa
    why is the actual band size different from the predicted?

  • Immunohistochemistry of Human Brain, cortex tissue at an antibody concentration of 5.0µg/ml using anti-TBX5 antibody (ab49308)

References

ab49308 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
Serum as blocking agent for 15 minute(s) · Concentration: 5% · Temperature: RT°C
Sample
Human Cell (Human pluripotent stem cell derived cardiomyocyte)
Specification
Human pluripotent stem cell derived cardiomyocyte
Permeabilization
Yes - saponin
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Aug 27 2014

Answer

Thank you for your question. The peptide immunogen for ab49308 has 100% homology to the Scyliorhinus canicula T-box protein 5 (Tbx5) sequence you provided. I hope this is helpful. Please contact me again if you have any further questions.  

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Answer

Thank you for your enquiry. Thus far I have found the following information regarding potential reactivity of our TBX5 antibodies to Scyliorhinus canicula: ab58253 - no chance for cross reactivity ab86370 - region of immunogen overlaps partially but this antibody is a monoclonal so about 50/50 chance for cross reactivity ab101227 - no chance for cross reactivity ab49308 - still waiting on information from our sourcing lab and will forward you any information I receive. I hope this is helpful. Please let me know if you have any further questions.

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Answer

Please find below two links to our recommended protocols: One details a full nuclear extraction protocol https://www.abcam.com/ps/pdf/protocols/Nuclear%20fractionation%20protocol.pdf one details the extraction using RIPA buffer:https://www.abcam.com/ps/pdf/protocols/WB-beginner.pdf I also enclose below the section on RIPA buffer preparation: RIPA buffer (Radio Immuno Precipitation Assay buffer) 150 mM sodium chloride 1.0% NP-40 or Triton X-100 0.5% sodium deoxycholate 0.1% SDS (sodium dodecyl sulphate) 50 mM Tris, pH 8.0 RIPA buffer is also useful for whole cell extracts and membrane-bound proteins, and may be preferable to NP-40 or Triton X100-only buffers for extracting nuclear proteins. It will disrupt protein-protein interactions and may therefore be problematic for immunoprecipitations/pull down assays. I hope this information helps, please do not hesitate to e-mail me back if you need further assistance and I will do my best to help you. Good luck with your experiments.

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Question

BATCH NUMBER 3509322 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE zebrafish embryos PRIMARY ANTIBODY human tbx5 antibody ( Abcam, only an different amino acid between human and zebrafish tbx5), 1/50,000, incubate in 4 degree celcius (overnight). Wash with TBST, 3 times for 10 minutes. DETECTION METHOD ABC reagent (Vectastain) was used for signal amplification, 30 minutes. Detection: 4-chloro-1-naphthol in 10ml methanol POSITIVE AND NEGATIVE CONTROLS USED GFP protein was successfully detected using this method. ANTIBODY STORAGE CONDITIONS -20 SAMPLE PREPARATION Cholate extraction buffer (10mM pH8.0 Tris, 2% cholic acid) with protease inhibitor cocktail, homogenised and and shake for overnight (4 degree celsius). Centrifuge and supernatant was collected. AMOUNT OF PROTEIN LOADED 10 ul ELECTROPHORESIS/GEL CONDITIONS SDS-PAGE: 12% separating gel + 4% stacking gel TRANSFER AND BLOCKING CONDITIONS transfer buffer (3.03g Tris-base, 14.4g glycine / litter), transfer for 2 hours 3% BSA as blocking agent 90 minutes. SECONDARY ANTIBODY Goat-Anti-Rabbit IgG-Peroxidase conjugated [another company] 1:5,000, incubates for 2 hours+ Wash with TBST, 3 times for 10 minutes. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes ADDITIONAL NOTES We are unable to detect tbx5 protein in 22hpf(hours post fertilization) wild type zebrafish embryos, It would be very kind, if you can suggest us which might be the error or causes that we can not detect tbx5 protein. Thank you very much.

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Answer

Thank you for your enquiry. I am sorry to hear you are having a problem with ab49308. As you mention, this antibody has so far not been tested in zebrafish but we would expect the antibody to work in this species due to the homology of the protein in human and zebrafish. I would like to suggest the following modifications to your protocol: -run a positive control along your tissue to make sure that your samples do contain significant levels of TBX5 protein. We recommend HepG2 whole cell lysate, which can be purchased from Abcam, reference ab7900. - use a RIPA lysis buffer to extract and concentrate the TBX5 protein in your samples. I am concerned that the current extraction protocol you are using is not optimal for nuclear protein extraction. Please let me know if you need the specific details of this lysis buffer, I will forward to you the protocol details. -make sure you load 20-30ug protein per well -please make sure your transfer buffer contains 20% methanol as it may be that the transfer is not working well currently. Check with a ponceau staining that the transfer has been optimal. -block the membrane with 5% milk in TBST as the antibody prefers milk blocking in our hands (typically 1 hour and incubate primary in TBST). -try different dilutions of primary antibody. We recommend a concentration of Use at a concentration of 5.0 µg/ml with HepG2 cell lysate, but it may be that you need to increase this a bit as it depends on the concentration of protein in the samples. I think a 1/50 000 dilution is currently too weak. -Finally, we recommend to use an ECl+ or more sensitive kits to detect the final results; I am concerned that the current detection method you are using is not suitable for this type of western blotting. Please let me know if this helps and do not hesitate to contact us for further advice. Best of luck with your research,

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