Overview

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

    Goat IgG whole molecule

  • Conjugation
    Biotin

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer
    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 0.42% Potassium phosphate, 0.88% Sodium chloride, 1% BSA
  • Concentration information loading...
  • Purity
    Affinity purified
  • Purification notes
    This product was prepared from monospecific antiserum by immunoaffinity chromatography using Goat IgG coupled to agarose beads followed by solid phase adsorption(s) to remove any unwanted reactivities.
  • Conjugation notes
    Biotinamidocaproate N-Hydroxysuccinimide Ester (BAC) Biotin/Protein Ratio: 10-20 BAC molecules per Rabbit IgG molecule
  • Clonality
    Polyclonal
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab6740 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
Dot blot
ELISA
IHC-P
IHC-Fr
Immunomicroscopy
ICC/IF
WB
  • Application notes
    Dot: Use at an assay dependent dilution.
    ELISA: Use at an assay dependent dilution.
    IHC-P: 1/100 (PMID 18930986).
    IHC-Fr: Use at an assay dependent dilution.
    IM: Use at an assay dependent dilution.
    WB: Use at an assay dependent dilution.


    Not yet tested in other applications.
    Optimal dilutions/concentrations should be determined by the end user.
  • References

    This product has been referenced in:
    • Costa GMJ  et al. Characterization of spermatogonial cells and niche in the scorpion mud turtle (Kinosternon scorpioides). Gen Comp Endocrinol N/A:N/A (2018). Read more (PubMed: 29966660) »
    • Cockrell AS  et al. A spike-modified Middle East respiratory syndrome coronavirus (MERS-CoV) infectious clone elicits mild respiratory disease in infected rhesus macaques. Sci Rep 8:10727 (2018). Read more (PubMed: 30013082) »
    See all 10 Publications for this product

    Customer reviews and Q&As

    1-2 of 2 Abreviews or Q&A

    Answer

    Thank you for your enquiry. This antibody has not been preadsorbed against rat tissue. In your case I would recommend ab7124 as this is a donkey polyclonal to goat IgG H&L (biotin-conjugated) pre-adsorbed antibody that has minimum cross reactivity to Chicken, Guinea Pig, Hamster, Horse, Mouse, Rabbit and Rat Serum Proteins. Using the rabbit antibody on rat tissue is very difficult because these species are very close and there is often high background. ab7124 should be better for your purposes. I hope this information helps, please do not hesitate to contact us if you need any more advice or information,

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    Question

    Thanks for your reply. Please see responses below. Apologies, but I don't have the PO number immediately available. The lot number is 48073. 1. Please describe the problem (high background, low signal, no signal etc). There does appear to be high background with the combination of ab1664, the ab6740 secondary, and a strepavidin-fluorophore from Pharmingen. I've tried strepavidin-PE, APC, TexasRed, etc. 2. On what material are you testing the antibody in FACS? •Species? •Cell type? •Cell line? I've been looking at epidermal cells from B6 mouse ear, gating on scatter characteristics, then CD45+ CD3+ (T-cells), and looking for CCR4 positive cells. 3. Which buffer did you use for cell suspension? We stain in FACS buffer, which is normal PBS + 2% FCS + 0.05% sodium azide. 4. How many cells did you use? ~1 million epidermal cells per sample. 5. Did you permeabilize the cells? How? No. 6. Primary antibody •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation time, wash steps (multiple short washes are more effective than fewer longer wash steps)? Dilution is 1 microliter of ab1664 per 50 microliters (the volume we stain in). We incubate for ~15 to 30 minutes. We then wash with FACS buffer (PBS + sodium azide + FCS), and then repeat the staining procedure with the secondary rabbit antibody and strepavidin+fluorophore tertiary. 7. Secondary antibody •What secondary antibody are you using? •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation, wash steps? •Do you know whether the problems you are experiencing come from the secondary? •What detection method are you using? ab6740, also dilution of 1 microliter of ab6740 per 50 microliters for a stain of one million cells, as above. Washing is as above. I then apply the strepavidin-fluorophore at the same dilution and incubate (again usually about 15 minutes). This is followed by a final wash. We usually FACS 1 million cells in a final volume of 200 microliters. 8. Which detection system did you use? •Detection wavelength The machine is a BD biosciences LSR 1. I've used a variety of fluorophores, corresponding to multiple wavelengths for FITC, PE, TexasRed, APC, and APC/Cy7. 9. Did you apply positive and negative controls along with the samples? Please specify. Negative controls has been splenocytes from normal B6 mice, which are expected to have cells that largely lack mCCR4. No positive controls have been really available. The strepavidin-fluorophores are known to work as they're widely used with a number of other biotinylated antibodies (admittedly many from Pharmingen). The rabbit anti goat secondary hssn't been tested because the only other goat antibody we have is ab1661 (anti-CCR10 and not advertised by Abcam as suitable for FACS. Been having the same issues with that one as well, but that's a different story). 10. Optimization attempts •How many times have you tried the FACS? •Do you obtain the same results every time? •What steps have you altered? Have tried the FACS 3 or 4 times. I've maintainly altered procedures for the collection of epidermal cells, but haven't really tried changing the FACS staining or acquisition steps.

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    Answer

    Thank you very much for your patience and for the details in which you have provided. I contacted the originator of ab1664 and below is the FACS protocl that they used. I hope this will be of use to you and if you continue to experience difficulty with this antibody please let me know and I will send you a replacement or a refund. FACS Protocol: 1. Pellet approx. 200,000 cells by centrifugation. 2. Resuspend the cell pellet in 50-75ul of medium with 5% FBS. 3. Add 1-5 ul of the anti-CCR antibody. 4. Incubate at 4 degree C for 45 minutes. 5. Wash the cells two or more times before adding secondary antibody. 6. Try using FITC conjugated F(ab’)2 anti goat antibody at appropriate dilution. If using anti-goat IgG then a blocking step indicated below is strongly recommended to reduce non-specific background. 7. Wash the cells and analyze. Controls and Blocking Steps: 1. Blocking of Fc-receptors: If one intends to use secondary IgG antibody raised in rabbits then the investigator initially should pre-incubate mouse cells with normal rabbit IgG or 2% heat inactivated normal rabbit serum for 30 minutes and use secondary rabbit antibody system at appropriate dilution with 100ug/ml of normal rabbit IgG. 2. Negative Control: Use another antigen affinity purified goat antibody at similar concentration to a protein that is not expressed on mouse cells as negative control.

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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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