Overview

  • Product name

    Anti-DDX3 (phospho T322) antibody
    See all DDX3 primary antibodies
  • Description

    Rabbit polyclonal to DDX3 (phospho T322)
  • Host species

    Rabbit
  • Specificity

    Detects DDX3 only when phosphorylated at threonine 322
  • Tested applications

    Suitable for: ELISA, ICC/IFmore details
  • Species reactivity

    Reacts with: Human
    Predicted to work with: Mouse, Rat
  • Immunogen

    Synthetic phosphopeptide derived from human DDX3 around the phosphorylation site of threonine 322 (V-A-TP-P-G).

Properties

Applications

Our Abpromise guarantee covers the use of ab61043 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
ELISA 1/5000.
ICC/IF 1/500 - 1/1000.

Target

  • Function

    ATP-dependent RNA helicase. Acts as a cofactor for XPO1-mediated nuclear export of incompletely spliced HIV-1 Rev RNAs. Also involved in HIV-1 replication. Interacts specifically with hepatitis C virus core protein resulting in a change in intracellular location.
  • Sequence similarities

    Belongs to the DEAD box helicase family. DDX3/DED1 subfamily.
    Contains 1 helicase ATP-binding domain.
    Contains 1 helicase C-terminal domain.
  • Cellular localization

    Nucleus speckle. Cytoplasm. Located predominantly in nuclear speckles and, at low levels, throughout the cytoplasm. Located to the outer side of nuclear pore complexes (NPC). Shuttles between the nucleus and the cytoplasm in a XPO1-dependent manner.
  • Information by UniProt
  • Database links

  • Alternative names

    • ATP dependent RNA helicase DDX3X antibody
    • ATP-dependent RNA helicase DDX3X antibody
    • CAP Rf antibody
    • DBX antibody
    • DDX14 antibody
    • DDX3X antibody
    • DDX3X_HUMAN antibody
    • DEAD (Asp Glu Ala Asp) box polypeptide 3 X linked antibody
    • DEAD (Asp-Glu-Ala-Asp) box helicase 3, X-linked antibody
    • DEAD box antibody
    • DEAD box protein 3 antibody
    • DEAD box protein 3 X-chromosomal antibody
    • DEAD box X isoform antibody
    • DEAD box, X isoform antibody
    • DEAD/H (Asp Glu Ala Asp/His) box polypeptide 3 antibody
    • DEAD/H box 3 antibody
    • DEAD/H box 3, X-linked antibody
    • Helicase like protein 2 antibody
    • Helicase-like protein 2 antibody
    • HLP2 antibody
    • X isoform antibody
    • X-chromosomal antibody
    see all

Images

  • 1/500-1/1000 ab61043 staining phosphorilated DDX3 on HuvEc cells treated with 20% serum for 30 minutes, only in the absence of the immunizing phosphopeptide.

References

ab61043 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Answer

Thank you very much for your answer. As my colleage is out of the office today, I am forwarding you already the answer from the laboratory in regards to the specificity of the ab61043. Please find the answer here below. I would like to reassure you again, that we do cover all our products with the guarantee to work as stated on the datasheet. If therefore the suggestions of my colleague will not help to resolve this issue, we will replace or refund this antibody for you. Can you please also confirm that the samples have been thoroughly denatured (with SDS and heat) and reduced for the loading?  Also, can you maybe send us an image of the results? Please find here the answer from the lab: " The immunogen peptide for this antibody has no significant homology with DEAD box domain. The name of the target protein (DEAD box protein) is causing confusion. (A)-This antibody was produced for phosphorylation site T323 of human DDX3X (We count phosphorylation site as T322, because the first M will be removed after the protein is synthesized).  (B)-Other name for this target is DEAD box protein 3.  (D)-By homologue search, the immunogen peptide only hit DDX3X.  (E)-The immunogen peptide is not located or has any significant homology in/to helicase ATP-binding domain, Q motif, and/or DEAD box, so this antibody will not react with any proteins containing these domains.  (F)-The reference for phosphorylation site: Biochem Biophys Res Commun. 2007 May 11;356(3):668-73. Epub 2007 Mar 15. " Please let me know how you are proceeding and whether you have any questions.  I wish you meanwhile a good weekend.

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Question

> 1) Abcam product code  ab61043 > > 2) Abcam order reference number or product batch number 403798 > > 3) Description of the problem: no band / high background (many non-specific bands) / wrong band size / other? > > Band is 100kda when it should be 72kda > > 4) Sample preparation: > > Type of sample (whole cell lysates, fraction, recombinant protein…): > > Species : Human > > Lysis buffer : > > 50mM HEPES pH 7.5(or Tris/HCl pH 7.5) > > 150mM NaCl > > 1mM EDTA > > 10% Glycerol > > 0.5% NP-40 > > > > Protease inhibitors: Yes > > Phosphatase inhibitors : Yes > > Reducing agent    :               DTT 2x sample buffer > > Boiling for ≥5 min? yes/no Yes > >  Protein loaded ug/lane or cells/lane :            25,000cells > > Positive control : > > Negative control : > > 5) Percentage of gel 10% > > Type of membrane  PVDF > > Protein transfer verified    Yes > > Blocking agent and concentration 5% Marvel > > Blocking time    1hr > > Blocking temperature Room Temp > > 6) Primary antibody (If more than one was used, describe in “additional notes”) : > > Concentration or dilution 1:500 > >  Diluent buffer  5% Marvel > > Incubation time Overnight > > Incubation temperature: 4 degrees > > 7)  Secondary antibody: > > Species: anti rabbit > > Reacts against: > > Concentration or dilution 1:1000 > > Diluent buffer  5%Marvel > > Incubation time 2 hrs > > Incubation temperature: Room Temp > > Fluorochrome or enzyme conjugate: HRP > > > > > > 8) Washing after primary and secondary antibodies: > > Buffer TBS-tween > > Number of washes               3 > > > > > > 9)Detection method > > ECL > > > > 10) How many times have you run this staining? Multiple times > > Do you obtain the same results every time?  Yes > > What steps have you altered to try and optimize the use of this antibody?

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Answer

Thank you for taking time to complete our questionnaire. Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab61043 : 1) I would suggest to use the antibody in a tested application, ELISA or ICC/IF. This antibody may not be suitable for Western Blot, we do not have any experimental data for this application. Our products are not covered by our 6 months Abpromise guarantee for untested applications (or untested species). For more details about the Abpromise guarantee, please visit the following link : www.abcam.com/abpromise 2) I would recommend to use BSA as blocking agent and dilution solution for both primary and secondary antibodies. Milk is not recommended for studies of phospho-proteins because milk contains casein which is a phospho-protein; it may cause background because the phospho-specific antibody detects the casein in the milk. I hope these suggestions are helpful.

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Answer

Thank you for contacting us. As far as we are aware, ab61043 has never been tested in western blot. All tested applications are specified on our datasheets, and these are updated as soon as any new information is brought to our attention. I am attaching our questionnaire so that we can gather further information regarding the samples tested and the protocol used. Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results. How different is the oberved molecular weight from the expected one? I look forward to receiving your reply.

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