Anti-5 Lipoxygenase / 5-LO (phospho S271) antibody (ab59395)

Overview

  • Product name
    Anti-5 Lipoxygenase / 5-LO (phospho S271) antibody
    See all 5 Lipoxygenase / 5-LO primary antibodies
  • Description
    Rabbit polyclonal to 5 Lipoxygenase / 5-LO (phospho S271)
  • Host species
    Rabbit
  • Specificity
    This detects endogenous levels of 5 Lipoxygenase only when phosphorylated at serine 271.
  • Tested applications
    Suitable for: WB, ELISA, IHC-Pmore details
  • Species reactivity
    Reacts with: Human
    Predicted to work with: Mouse
  • Immunogen

    Synthetic peptide within Human 5 Lipoxygenase/ 5-LO (phospho S271). The exact sequence is proprietary.

  • Positive control
    • IHC-P: Human skeletal muscle tissue. WB: Extracts from HuvEc cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab59395 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
WB 1/500 - 1/1000. Detects a band of approximately 70 kDa (predicted molecular weight: 78 kDa).
ELISA 1/10000.
IHC-P Use at an assay dependent concentration.

Target

  • Function
    Catalyzes the first step in leukotriene biosynthesis, and thereby plays a role in inflammatory processes.
  • Pathway
    Lipid metabolism; leukotriene A4 biosynthesis.
  • Sequence similarities
    Belongs to the lipoxygenase family.
    Contains 1 lipoxygenase domain.
    Contains 1 PLAT domain.
  • Post-translational
    modifications
    Serine phosphorylation by MAPKAPK2 is stimulated by arachidonic acid. Phosphorylation on Ser-523 by PKA has an inhibitory effect. Phosphorylation on Ser-272 prevents export from the nucleus.
  • Cellular localization
    Cytoplasm. Nucleus matrix. Nucleus membrane. Shuttles between cytoplasm and nucleus. Found exclusively in the nucleus, when phosphorylated on Ser-272. Calcium binding promotes translocation from the cytosol and the nuclear matrix to the nuclear envelope and membrane association.
  • Information by UniProt
  • Database links
  • Alternative names
    • 5 Lipoxygenase antibody
    • 5 LO antibody
    • 5 LOX antibody
    • 5-lipoxygenase antibody
    • 5-LO antibody
    • 5-LOX antibody
    • 5LOX antibody
    • 5LPG antibody
    • ALOX 5 antibody
    • Alox5 antibody
    • Arachidonate 5 lipoxygenase antibody
    • Arachidonate 5-lipoxygenase antibody
    • arachidonic 5-lipoxygenase alpha-10 isoform antibody
    • arachidonic 5-lipoxygenase delta-10-13 isoform antibody
    • arachidonic 5-lipoxygenase delta-13 isoform antibody
    • arachidonic 5-lipoxygenase delta-p10 isoform antibody
    • Arachidonic acid 5 lipoxygenase antibody
    • Leukotriene A4 synthase antibody
    • LOG 5 antibody
    • LOG5 antibody
    • LOX5_HUMAN antibody
    • MGC163204 antibody
    see all

Images

  • All lanes : Anti-5 Lipoxygenase / 5-LO (phospho S271) antibody (ab59395) at 1/500 dilution

    Lane 1 : Extracts from HuvEc cells.
    Lane 2 : Extracts from HuvEc cells plus phosphopeptide.

    Predicted band size: 78 kDa
    Observed band size: 70 kDa
    why is the actual band size different from the predicted?

References

ab59395 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

Question

DESCRIPTION OF THE PROBLEM Non-specific band SAMPLE mouse lung whole cell lyasate PRIMARY ANTIBODY Concentration or dilution : 1:500 Diluent buffer : 0.1% TBS-Tween Incubation time : overnight Incubation temperature: 4℃ Washing Buffer 0.1% TBS-Tween Number of washes 5 X 5 mins DETECTION METHOD using LAS-1000 (Fuji) POSITIVE AND NEGATIVE CONTROLS USED negative control : normal mouse lung cell ANTIBODY STORAGE CONDITIONS -20 ℃ SAMPLE PREPARATION Lysis buffer : novagen phophosafe reagent Protease inhibitors: added Reducing agent: added (2-ME and DTT) Boiling for ≥5 min? yes/no : no AMOUNT OF PROTEIN LOADED Protein loaded ug/lane or cells/lane : 60ug/lane ELECTROPHORESIS/GEL CONDITIONS 10% SDS-PAGE Gel TRANSFER AND BLOCKING CONDITIONS Type of membrane : PVDF (pore size :0.45 um) Protein transfer verified : verify using pre-stained protein marker Blocking agent and concentration : 5% non-fat milk for non-phosphorylated type and 5% BSA for phosphorylated type protein Blocking time: 1 hr Blocking temperature : RT SECONDARY ANTIBODY Species: goat Isotype: IgG Reacts against: rabbit IgG Concentration or dilution : 1:2000 Diluent buffer : 0.1% TBS-Tween Incubation time : 1.5 hrs Incubation temperature: RT Fluorochrome or enzyme conjugate: HRP Washing Buffer 0.1% TBS-Tween Number of washes 5 X 5 mins HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? I altered Ab concentration, incubation time and temperature ADDITIONAL NOTES I would like to confirm which bands on blotted membranes are my target protein. Size (molecular weight) of band is different from that of datasheet

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Answer

Thank you for contacting us. The mouse 5-lipoxygenase, like the human protein is 77.9 kDa. You would therefore expect to see a band at around this size. However, this size can vary considerably depending on the cell lysate produced, how the gel is performed and the buffers used. You appear to have specific bands from both antibodies appearing at around 60kDa. With a non-specific band with the Phospho-5-Lipoxygenase at around 45 kDa. In your protocol you mention that you used both DTT and b-mercaptoethanol. This is usually not necessary and it should be sufficient to use either DTT (final concentration of 20 mM) or b-mercaptoethanol (final concentration 5%). Additionally, unless you are attempting to run a native gel I would suggest heating the samples for 5 minutes at 95-100°C to allow the proteins to denature. You do not mention which buffers were used but the ones we typically recommend can be found here: https://www.abcam.com/index.html?pageconfig=resource&rid=11375 Additionally in order to check if the antibody is specifically recognising the phosphorylated form of the protein you could de-phosphorylate the protein as described here: https://www.abcam.com/index.html?pageconfig=resource&rid=11407 I would also strongly recommend carrying out a no primary control to ascertain if this is contributing to the specificity. I hope this information has been of help to you, if you remain unsatisfied with the performance of this antibody please let me know.

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

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