Overview

  • Product name

  • Description

    Rabbit polyclonal to Flagellin
  • Host species

    Rabbit
  • Specificity

    This antibody is specific for bacterial Flagellin (FliC).
  • Tested applications

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

    Reacts with: Escherichia coli, Salmonella anatum, Salmonella selandia
  • Immunogen

    Purified recombinant Flagellin made in BL21 bacteria.

  • Positive control

    • Lysates transfected with purified flagellin, E. Coli total lysate or 293T transfected with recombinant protein.

Properties

Applications

Our Abpromise guarantee covers the use of ab93713 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 1/10000 - 1/20000. Detects a band of approximately 60 kDa.
ICC/IF 1/100.

Target

  • Relevance

    Flagellin (FliC) is a subunit protein that polymerizes (along with other proteins) to form the filaments of bacterial flagella. Assembly of the bacterial flagellum occurs in a precise, temporal order where the basal component (FlgE, FlgK, and FlgL are assembled inside the bacterial membrane, followed by exportation of the filament cap protein FliD, and secretion of about 20,000 flagellin monomers (FliC) through the channel. FliC monomers are polymerized to form the tail filament. FliC monomers can function as pathogen-associated molecular patterns (PAMPs), and can be detected by host cells through surface-localized toll-like receptor 5 (TLR5) and cytosolic Nod-like receptors (NLRs).
  • Cellular localization

    Secreted. Bacterial flagellum.
  • Database links

    • Alternative names

      • 41 kDa antigen antibody
      • b1923 antibody
      • flaF antibody
      • Flagellar filament 41 kDa core protein antibody
      • Flagellin antibody
      • fliC antibody
      • hag antibody
      • JW1908 antibody
      • P41 antibody
      see all

    Images

    • All lanes : Anti-Flagellin antibody (ab93713) at 1/10000 dilution

      All lanes : 293T transfected with recombinant protein

      Observed band size: 60 kDa
      why is the actual band size different from the predicted?

    • Ab93713 (1:100) staining Flagellin in bone marrow-derived macrophages infected with salmonella.

      Flagellin primary antiserum followed by Alexa488 secondary (green). Actin (red) stained with AlexaFluor 594-phalloidin. Nuclei stained with DAPI.
    • Ab93713 (1:100) staining Flagellin in bone marrow-derived macrophages infected with salmonella.

      Flagellin primary antiserum followed by Alexa488 secondary (green). Actin (red) stained with AlexaFluor 594-phalloidin. Nuclei stained with DAPI.

    References

    This product has been referenced in:

    • Barnowski C  et al. Advantages and Limitations of Integrated Flagellin Adjuvants for HIV-Based Nanoparticle B-Cell Vaccines. Pharmaceutics 11:N/A (2019). Read more (PubMed: 31052410) »
    • Zervos C  et al. Immunoglobulin G from single plasma donor in immune globulin intravenous causes false positive pyrogen test. Biologicals 59:12-19 (2019). Read more (PubMed: 31023510) »
    See all 8 Publications for this product

    Customer reviews and Q&As

    1-9 of 9 Abreviews or Q&A

    Answer

    Thank you for contacting us.

    Unfortunately, I do not know which bacteria you are investigating. Ifyou are looking at peritrichous bacteria such as E.coli or salmonella, the flagella are all over the bacteria, so the antibody is going to light up the whole cell. Thus,I am not sure whether the antibodywill be able toshow what you want it to.

    Having said this though, I am more than happy to investigate this case further for you and see if there is anything we can do. In order to proceed with this, I have enclosed a questionnaire and I would appreciate if you could complete this. It will help you put the information we require together very easily. I would appreciate if you could also provide an image which would help us to assess the results.

    Thank you for your time and cooperation. We look forward to receiving the completed questionnaire.

    Read More
    Application
    Western blot
    Sample
    Salmonella typhi Cell lysate - other (Flagellin preparation of whole cell cultures)
    Gel Running Conditions
    Reduced Denaturing (12% SDS-Page gel)
    Loading amount
    100 µg
    Specification
    Flagellin preparation of whole cell cultures
    Blocking step
    Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

    Amanda Gonzales

    Verified customer

    Submitted Feb 28 2018

    Answer

    Thank you for getting in touch.


    Please find below the protocols for infecting macrophages with salmonella and the protocol for staining the infected macrophages.


    I wish you successful experiments.


    Salmonella intracellular replication protocol


    Day 1:


    1. Seed macrophages in triplicate (3 wells per data point) into 24 well dishes at a density of 3 x 105 cells/well


    2. Inoculate cultures (3 mL of LB + appropriate antibiotics*). Grow O/N at 37 °C with aeration.


    Day 2:


    1. O/N bacterial cultures should be very turbid. Dilute 1:10 into PBS, measure OD600 and calculate number of bacteria necessary for infection. (Blank with 1:10 LB in PBS). OD600 of 1 typically is 1 x 109 cfu/mL, different spectrophotometers vary, so it is important to establish the appropriate number the first several times an assay is done, to ensure that the actual MOI is correct.


    2. Remove amount of bacteria necessary for infection, add normal mouse serum to final concentration of 20%, opsonize by shaking at 37 °C for 15-20 minutes (not longer than 30 though, because otherwise the bacteria will grow and your MOI will be off). Do everything in a total volume of no more than 1 mL.


    3. Wash bacteria 3x with PBS, vortex well while washing – the serum causes the bacteria to clump.


    4. After final wash and spin, bring up bacteria in appropriate volume to ensure that 100 uL of bacteria is an MOI of 10.


    5. Add bacteria to appropriate wells of 24-well plate, spin in table-top centrifuge at 1000 rpm for 5 minutes.


    6. Place in 37 °C humidified incubator for 60 min.


    7. Add hi conc. gentamycin (100 mg/ml stock à 100 ug/mL final). This will kill extracellular bacteria without affecting survival of intracellular bacteria. Incubate cells with gentamycin for 60-90 minutes.


    8. Aspirate medium, wash 2x with DMEM, add back 1 mL of DMEM + lo conc. gentamicin (10 ug/mL final). At this timepoint, one set of cells can be harvested** to determine amount of bacteria that actually made it inside. This is considered T0.


    9. Timepoints can be harvested whenever after that. People typically harvest somewhere between 8-12 hours, and again at 24 hours. When harvesting later timepoints, harvest supernatant as well as cells, and combine. Approximately 10-20% of total bacteria are in supernatant at 24 hour timepoint.


    * Antibiotics (all at 1000x):


    Streptomycin: 100 mg/mL stock


    Kanamycin: 40 mg/mL stock


    Chloramphenicol: 25 mg/mL stock


    Tetracycline 10 mg/mL


    The bacterial strain background that we use is SL1344. This is a pathogenic strain of Salmonella enterica, serovar Typhimurium, originally isolated from an infected cow. It is a naturally occurring rpsL mutant, and is therefore resistant to streptomycin. This can be useful when culturing bacteria out of infected organs.


    ** Harvesting of timepoints:


    1. Remove supernatant from cells. (When harvesting later timepoints, spin supernatant at max speed for 2 minutes, aspirate post-spin sup. You may or may not see a small pellet at this point, which is cell debris plus some bacteria).


    2. Add 200 uL of 1% Triton X-100 in PBS to wells. Incubate 5’ at RT to allow cell lysis.


    3. Pipette up-and-down thoroughly but not too vigorously if you can avoid it (it will be very foamy), harvest into eppendorf (at later timepoints, harvest into eppendorf containing pellet from step 1, and pipette several times to resuspend/lyse pellet). Add 800 uL of DMEM to empty well, pipette up-and-down several times, combine with 200 uL of lysed cells. Vortex to mix, prepare 10-fold serial dilutions.


    4. Plate appropriate dilutions onto appropriate antibiotic-containing plates.


     


    Staining the infected macrophages


    All steps at Room Temp (RT)


    Fix 4% PFA in PBS for 15 min at RT


    Wash 2x with PBS + 1% BSA


    Block with Fc block in PBS +  1% BSA for 30 min RT.


    Block with perm buffer: 0.1% saponin in PBS + 1% BSA for 1hr at RT


    Wash 1× with perm buffer (0.1 % saponin in PBS + 1% BSA) RT


    Stain in perm buffer:


    1hr RT for primary antibody (The titer of antibody must be empirically determined.) and 1hr for secondary antibody. Make sure that secondary antibody does not cross-react with Rat as Fc block is made in rat.


    Wash 2x with perm buffer


    Wash 1x with PBS 


    Mount on slides.

    Read More

    Answer

    Thank you for contacting us.

    The flagellin antibody was generated against recombinant purified
    full-length FliC from E Coli DH5a. There are similarities at the N and C
    termini with P aeruginosa FliC but it is probably not enough for the
    antibody to recognize it. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

    Read More

    Answer

    Thank you for contacting us and thank you for your patience.

    The protocol seems fairly standard. However, she was in contact with the originator of this antibody, and here aretheir thoughts:

    1) The rods are certainly full of newly expressed Flagellin not yet assembled in the tail, which is probably why not only flagellae but whole rods are stained

    2) If Flagellin monomers are still polymerised to form the tail, it is possible that the protein's structure is compromised and thus a detection by the antibody fails



    I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

    Use our products? Submit an Abreview. Earn rewards!
    https://www.abcam.com/abreviews

    Read More

    Question
    Answer

    Thank you for your call earlier and for your patience while I have looked into your enquiry.

    Based on the sequences available on UniProt, I don't expect any of our flagellin antibodies to react with pseudomonas samples. There were multiple putative sequences available for the proteins, but I've listed the range of BLAST results below.

    Ab106146: 29-38% identity between immunogen and pseudomonas
    Ab93713: 35-51% identity
    Ab127983: 29% identity

    I am sorry that we don't have an antibody that might react with your species. I would recommend looking on http://www.biocompare.com for a high throughput search of antibodies to this target.

    Please let me know if you have any questions, or if there is anything else that we might be able to do for you.

    Read More

    Answer

    Thank you for contacting Abcam. The concentration of protein in ab93713 is between 30 and 40 mg/ml. We do not know how much of that total is made up with antibody because the sample is serum, not purified antibody. Please let me know if there is anything else I can help you with.

    Read More

    Answer

    Thank you for contacting us. The total protein concentration of ab93713, lot GR35089-1, is around 30-40 mg/ml. However, since this is whole serum containing a wide variety of proteins, the concentration of the specific anti-Flagellin antibody cannot be determined. Please be reassured that this antibody is covered by our Abpromise and we guarantee it to work in the tested species and applications as stated on the datasheet. Please find the recommended starting dilutions in the application notes on the datasheet. In the event that it is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note, free of charge replacement or refund. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

    Read More

    Question
    Answer

    J'ai été informé par laboratoire que l'anticorps ab93713 peut en effet détecter la flagelline de Salmonella typhimurium. Malheureusement ils n'ont jamais testé la réactivité de l'anticorps avec bacilis subtilis. Comme expliqué par téléphone, nous n’offrons pas d’échantillon gratuit à des fins de tests préliminaires. Cependant, si vous souhaitez tester ab93713 chez Bacillus subtilis, il me sera possible de vous offrir une remise. Après soumission d’une Abreview avec vos résultats, une remise correspondant à la valeur d’achat de ab93713 pourra être utilisable lors d’une prochaine commande d’anticorps primaire. Etapes à suivre : 1. Nous confirmer que vous souhaiteriez tester ab93713 chez Bacillus subtilis afin de recevoir le code de remise correspondant. Ce code doit être édité avant l’achat de ab93713 2. Acheter ab93713 par téléphone, fax ou internet (www.abcam.com) 3. Le tester 4. Nous faire part de vos résultats, positifs ou négatifs, grâce à notre système Abreview. Pour plus d’informations https://www.abcam.com/abreviews 5. Après soumission de votre Abreview, appeler notre service clientèle afin de passer votre prochaine commande grâce au code de réduction. Ce code de réduction est utilisable 120 jours après son édition et utilisable lors de l’achat d’un autre anticorps primaire. Même si ab93713 ne fonctionne pas chez Bacillus subtilis, après soumission de votre Abreview, vous pourrez commander gratuitement un anticorps primaire de notre catalogue. N’hésitez pas à nous demander plus d’informations concernant cette offre promotionnelle. Termes et Conditions : www.abcam.com/collaborationdiscount.  

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