Goat Anti-Rabbit IgG H&L (Alkaline Phosphatase) (ab6722)

Submit a review Q&A (6)References (15)

Overview

  • Product name
    Goat Anti-Rabbit IgG H&L (Alkaline Phosphatase)
    See all IgG secondary antibodies
  • Host species
    Goat
  • Target species
    Rabbit
  • Tested applications
    Suitable for: IHC-P, WB, ELISA, Dot blot, ICC/IF, IHC-Fr, ICCmore details
  • Immunogen

    Rabbit IgG whole molecule

  • Conjugation
    Alkaline Phosphatase

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C.
  • Storage buffer
    pH: 8.00
    Preservative: 0.1% Sodium azide
    Constituents: 0.00136% Zinc chloride, 0.0095% Magnesium chloride, 0.79% Tris HCl, 50% Glycerol, 0.87% Sodium chloride, 1% BSA
  • Concentration information loading...
  • Purity
    Affinity purified
  • Purification notes
    This product was prepared from monospecific antiserum by immunoaffinity chromatography using Rabbit IgG coupled to agarose beads followed by solid phase adsorption(s) to remove any unwanted reactivities.
  • Conjugation notes
    Alkaline Phosphatase (Calf Intestine) (Molecular Weight 140,000 daltons)
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab6722 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 at an assay dependent dilution.
WB 1/1000 - 1/4000.
ELISA 1/3000 - 1/5000.
Dot blot Use at an assay dependent dilution.
ICC/IF 1/200 - 1/1000.
IHC-Fr Use at an assay dependent dilution.
ICC Use at an assay dependent dilution.

Images

  • Western blot - Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (AP) (ab6722)
    Western blot - Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (AP) (ab6722)

    ab6722 was used at dilution 1/10000 with the primary antibody ab16731 in WB. See the review on ab16731.

  • ELISA - Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (AP) (ab6722)
    ELISA - Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (AP) (ab6722)

    ab6722 was used with the primary antibody ab2594 in ELISA. See Abreview on ab2594.

  • Western blot - Goat Anti-Rabbit IgG H&L (Alkaline Phosphatase) (ab6722)
    Western blot - Goat Anti-Rabbit IgG H&L (Alkaline Phosphatase) (ab6722)

    Western Blot of Anti-Rabbit IgG (H&L) (GOAT) Antibody.

    Lane M: 3 µl Molecular ladder.

    Lane 1: Rabbit IgG whole molecule.

    Lane 2: Rabbit IgG F(ab) Fragment.

    Lane 3: Rabbit IgG F(c) Fragment.

    Lane 4: Rabbit IgM Whole Molecule.

    Lane 5: Normal Rabbit Serum.

    All samples were reduced. Load: 50 ng per lane. Blocked for 30 min at RT.

    Primary Antibody: Anti-Rabbit IgG (H&L) (GOAT) Antibody 1:1,000 for 60 min at RT. Secondary antibody: Anti-Goat IgG (DONKEY) Peroxidase Conjugated Antibody 1:40,000 for 30 min at RT.

    Predicted/Obsevered Size: 25 and 50 kDa for Rabbit IgG and Serum, 25 kDa for F(c) and F(ab), 70 and 23 kDa for IgM.

    Rabbit F(c) migrates slightly higher.

  • Dot Blot - Goat Anti-Rabbit IgG H&L (Alkaline Phosphatase) (ab6722)
    Dot Blot - Goat Anti-Rabbit IgG H&L (Alkaline Phosphatase) (ab6722)

    Dot Blot of ab6722.

    Antigen: Rabbit IgG.

    Load: Lane 1 - 200ng; Lane 2 - 66.7ng; Lane 3 - 22.2ng; Lane 4 - 7.4ng; Lane 5 - 2.5ng.

    Secondary antibody: ab6722 used at 1:1,000 for 60 min at RT. Blocked for 60 min at RT.

    Reaction visualized using alkaline phosphatase substrate for 30 seconds at RT.

  • Western blot - Goat Anti-Rabbit IgG H&L (Alkaline Phosphatase) (ab6722)
    Western blot - Goat Anti-Rabbit IgG H&L (Alkaline Phosphatase) (ab6722)

    Lane 1: Rabbit IgG.

    Load: 100 ng per lane.

    Secondary antibody: ab6722 used at 1:1,000 for 60 min at RT. Blocked for 30 min at RT.

    Predicted/Observed size: 55 and 28 kDa, 55 kDa for Rabbit IgG.

References

This product has been referenced in:
  • Jang J  et al. Bioelectrochemical conversion of CO2 to value added product formate using engineered Methylobacterium extorquens. Sci Rep 8:7211 (2018). Read more (PubMed: 29739951) »
  • Liu CT  et al. Production of interleukin-4 in CD133+ cervical cancer stem cells promotes resistance to apoptosis and initiates tumor growth. Mol Med Rep 13:5068-76 (2016). WB . Read more (PubMed: 27121303) »
See all 15 Publications for this product

Publishing research using ab6722? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-6 of 6 Abreviews or Q&A

Question

Do I need to add a stop solution (ie NaOH) at the end of the sandwich ELISA after the colour develops with ALP chromogen or that is not necessary?

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Answer

Thank you for contacting us.

Just as with a common indirect ELISA it is important to stop the reaction using the appropriate stop solution.

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

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Question

I repeated the experiment and diluted plasma samples out to 1:4800 fold but this dilution dependent signal increase phenomenon persisted. In other words, the signal from the 1:4800 fold dilution was higher than the 1:2 dilution! I really doubt if this is a hook effect as I would expect the signal to decrease again after adequate dilution and not continue to rise. Then I thought whether it could be non-specific binding of detection ab (I used ab6722 - a Goat anti-rabbit ab because my primary is Rabbit anti-human). So I included controls in which I added only the secondary ab to plasma-coated wells, without any primary. I saw moderate signal from these wells (ie higher than BSA negative controls but weaker than corresponding plasma-coated wells with both primary and secondary added). Does this mean the secondary ab is binding non-specifically to something in the human plasma? Are you aware of cross-reacitivity of this Goat anti-rabbit ab (ab6722) with any human Ig??

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Answer

I am not aware of any cross reactivity of this antibody with human Ig. However we do offer a number of secondary antibodies which are designed to reduce background levels by either being pre-absorbed against other proteins and Igs or by digestion into F(ab) and Fc fragments. I've included links to a few of those below.

That said I do not feel that the background you are experiencing should be the cause for the type of response you have been seeing. This is usually something that can be easily subtracted from your results if you run 2 or 3 wells with this control as a baseline.

Another possibility is that it could be that the dilution buffer you are using is somehow causing this reaction. What buffer are you using? Have you tried using filtering this or using another buffer?

I hope that this information is helpful. Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.

These are links to the antibodies that have been pre-absorbed:

https://www.abcam.com/Goat-polyclonal-Secondary-Antibody-to-Rabbit-IgG-H-L-Alkaline-Phosphatase-pre-adsorbed-ab97072.html

https://www.abcam.com/Goat-polyclonal-Secondary-Antibody-to-Rabbit-IgG-H-L-Alkaline-Phosphatase-pre-adsorbed-ab97072.html

https://www.abcam.com/Goat-F-ab-2-polyclonal-Secondary-Antibody-to-Rabbit-IgG-H-L-AP-pre-adsorbed-ab98469.html

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Question

Many thanks for your reply. I really appreciate your suggestions. My detection is working well now. However I am facing some strange results which I would love your thoughts on please:   I am struggling to interpret an indirect ELISA I am making. It involved coating wells with human plasma overnight, blocked with milk the next day, then primary ab (Abcam), then detection ab (Abcam) and finally the ALP substrate. I ran parallel wells in which I coated with   1)      known concentration of antigen which is a recombinant protein to generate my standard curve and 2)      BSA (negative control) I got nice reproducible standard curves (R2=0.99) and uniformly negative signals from BSA. So all good. However I am seeing a dose-dependent increase in signal in plasma samples. As plasma dilution increased, signal increased - in other words, the more dilute the plasma (so in theory lesser amount of antigen) - the stronger the signal! I could not understand why. I repeated the experiments and I am seeing it every time (4x now). At first I thought it could be due to primary ab binding to "unoccupied sites in the wells" directly, which then generated a signal when secondary added, which would explain why the signal is stronger in diluted sample. But if that's the case I should see that in the BSA wells, but I am not. I did block the wells with milk after coating to ensure all unbound sites are occupied. Then I thought could it be something in the plasma which is inhibiting the substrate reaction (ALP). So the more dilute the plasma, the less the inhibition, and the stronger the signal. But plasma was only used to coat the plate overnight. The plates were then washed after every step with TBS-tween (2-4 times) after every step. So I would imagine not much of any “inhibitors” are around by the time substrate is added. Then I thought whether it could be a “hook” effect. In other words, the plasma is too concentrated and antigen are crowding up thus reducing the amount of binding. But I thought usually plasma then need to be diluted up to 100 folds to relieve the hook. But I am seeing a “nice” dose dependent increase in signal as plasma was diluted from 1:2, 1:4, 1:8 to 1:16 only.

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Answer

Thank you for contacting me again. It is always a pleasure to hear from you.

After reviewing your email, I am lead to believe that this really is a hook effect as you had mentioned. I am not an expert on this hook effect in plasma specifically, and so cannot comment on the 100 fold dilutions usually required, but your description does sound like a hook effect. These should eventually tail off with further dilutions.

The hook effect may also be created by overcoating with primary antibody; you may wish to further dilute your primary to tackle this as well.

I hope these suggestion help. Let me know if you have any questions.

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Question

Thanks for your advice and suggestions last time. I got another ab7468 and it is working well, both for WB and my reattempt ELISA.   However I have a question which I hope you may have an answer to:   After adding the pNPP substrate, I waited for 30 min, as recommended, but the signal was very weak. OD was around 0.1 and negative control 0.06. However as I waited for longer, up to 1.5 hour, the signal intensified in the samples and went up to about 0.4. Negative control still remained low at 0.06, which was good. The resultant standard curve was great (R2=0.99). However I could not understand why it took almost 60-90 min before the OD got up to a “good signal”. I don’t think I am simply increasing background as the standard curve was good and the longer duration did not intensify signal from any of the negative controls.   Is this longer than usual duration due to 1)      Inadequate primary ab? 2)      Inadequate secondary ab? 3)      Inadequate coating antigen on plate??  

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Answer

Thank you for contacting us, I am glad to hear that the new product is working well for you.

Usually when color is developing slowly it is one of three issues. 1) the plates and reagents have not come to room temperature. 2) the conjugates are weak, try to prepare these right before use and at correct concentration 3) the presence of peroxidases, azide or other contaminants are interfering with the reaction, these may be removed by dyalisis, gel filtration or with purification columns.

I hope that this information is helpful. Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.

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Question

I recently purchased your ab (ie Rabbit anti-human FNDC5 IgG), to be used in a ELISA. I am wondering what detection system would you recommend?   Ie According to the Abcam ELISA protocol, one option is HRP.   So Do I add a secondary ab conjugated (ie anti-rabbit IgG Fc, HRP conjugated), followed by washing and then dispensing 100 ul of substrate solution (ie TMB?)   Or would you recommend other secondary/substrate solution system?  

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Answer

Thank you for contacting Abcam.

Enzymatic detection with an antibody that has been conjugated to HRP or to AP is a widely used and robust method. As this target is located on the peroxisome membrane I would recommend using anAlkaline Phosphataseconjugated secondary for this target prehaps ab6722,Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (AP). Followed by an appropriate substrate detection of your choice. I recommend theAlkaline Phosphatase chromogen (BCIP/NBT). We do offer this as a ready to use product, ab7468.

I hope you have found this information helpful. Please let me know if you have any questions. I have places links to each of the products I have mentioned here below.

Ab6722:https://www.abcam.com/Goat-polyclonal-Secondary-Antibody-to-Rabbit-IgG-H-L-AP-ab6722.html

Ab7468:https://www.abcam.com/Alkaline-Phosp

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Question

I would like to receive a vial of ab34767 as a refund for my order for ab28664. Also, this is my purchasing code for another antibody I have ordered and that does not work with the replacement product you will send me :Po-IG/101857, order on the 18th August 2011 and for which I would like a refund or credits.   Please advise me how quickly the replacement antibody will arrive, as I would like to proceed with my experiments.   Thank you for your assistance!

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Answer

Thank you for your reply. I have sent a free vial of ab34767 to replace ab28664. I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. 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 have also issued a credit note for ab6722. The credit note may be used in one of the following ways: (1) Redeemed against the original invoice if this hasn't already been paid. (2) Held on the account for use against a future order. (3) A full refund can be offered where no other invoices are outstanding. Please contact your Finance department to confirm how you would like the credit note to be used and ensure it is not redeemed without your knowledge. To specifically receive a refund please ask your Finance department to contact our Finance department at creditcontrol@abcam.com or by telephone using the information at the “Contact Us” link in the top right corner of our website. The credit note ID is for your reference only, please refer to the credit note ID in any correspondence with our accounting department. We will send you the completed credit note by email or postal mail with the actual credit note number which will start with the letters CGB. I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service should you require further expert advice

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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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