Overview

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

    Goat 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: 0.878% Sodium chloride, 1% BSA, 0.788% Tris HCl, 0.0095% Magnesium chloride, 0.0014% Zinc chloride, 50% Glycerol
  • 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.
  • 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 ab6742 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 Use at an assay dependent concentration.
ELISA Use at an assay dependent dilution.
ICC/IF 1/200 - 1/1000.
WB Use at an assay dependent dilution.
IHC-P Use at an assay dependent concentration.
IHC-Fr Use at an assay dependent concentration.

Images

  • Ab6742 was used at dilution 1/4000 with the primary antibody ab20773 in ELISA. See the review on ab20773.

References

This product has been referenced in:
  • Hughes AJ & Herr AE Microfluidic Western blotting. Proc Natl Acad Sci U S A : (2012). WB . Read more (PubMed: 23223527) »
  • Takayama H  et al. High-level expression, single-step immunoaffinity purification and characterization of human tetraspanin membrane protein CD81. PLoS ONE 3:e2314 (2008). ELISA . Read more (PubMed: 18523555) »
See all 2 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Question
Answer

Thank you for contacting us.

As discussed we have many anti goat antibodies that can be used in ELISA and ICC/IF.

Idealy enzyme conjugated antibodies e.g. HRP, Alkaline phosphatase are the best suitable antibodies in ELISA. However these will not work in Immunofluorescence unless further steps in protocol applied.

We have FITC, TRITC or Cy5 conjugated antibodies in catalogue that can work in Immunofluorescence and Fluorescence ELISA.

Another option is choosing Biotin conjugated secondary antibody and then buying strepavidin or streptavidin conjugated HRP (ab7403).

ab5753, ab6740, ab6884, ab7124, ab7131 coul dbe a good option.

I know it sounds complicated however this is only way to combine ELISA and ICC/IF with one secondary antibody.

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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Answer

Thank you for your enquiry. I see no reason why you can't substitute in a different secondary antibody, but you want to consult the instructions of the kit that you have, or are interested in purchasing. If you have any more questions, please contact us again.

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Question

I have already sent a picture with details showing the latest attempt to get this antibody to work. But I will try to answer these questions and send the picture again. 1. Please describe the problem (high background, wrong band size, more bands, no band etc). There doesn't appear to be a correct or distinguishable band in the area that PI31 is supposed to be. Since the control is not working, I have no idea if my samples work or not, plus there is an incredible amount of background. My blots don't give me any usable data, and I don't know if it's because that protein may not be present in my samples (it should be), or that the primary antibody or something else is not working. 2. On what material are you testing the antibody in WB? •Species? •Cell extract/ Nuclear extract? •Purified protein? •Recombinant protein? I am testing the antibody with your control (ab7920), which doesn't give me a reaction. Then my samples are rat skeletal muscle extracts (homogenized tissue). I have also tried, kidney lysates and HeLa cell lysates with no reaction. 3. How much protein did you load? •How did you prepare the lysate for the analysis (protease inhibitors etc)? •Did you heat the samples? I have been trying to do a curve with my samples, so the load has been from 2-40 ug. Samples are not boiled, but samples are diluted in SDS reducing buffer which contains, beta-mercaptoethanol, glycerol, Tris, SDS, bromthymol blue. The human kidney control was not boiled, and arrived in SDS buffer, so not diluted either. 4. Primary Antibody •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation time, wash step? I used ab3893, lot 15623, which was raised in goat. I have used the recommended dilution of 0.5-2ug/ml, incubated at room temp. for 1-2 hours. Wash was atleast 3 times for 5 minutes each. 5. Secondary Antibody •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation time, wash step? •Do you know whether the problems you are experiencing come from the secondary? I used a bovine-anti-goat, and then your rabbit-anti-goat (ab6742), both are Alkaline phosphatase. I have used them at 1:3000 - 1:15,000 (normal secondary diln. for skeletal muscle is 1:5000). I have tried secondary only with several diln's as well. As the blot picture shows, the secondary only(ab6742) doesn't look good either. Again, incubation is 1 hour room temp, the same wash steps as before. 6. What detection method are you using? Alkaline phosphatase 7. Background bands •Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary?control) •Is the blocking step sufficient? (We recommend blocking the membrane by adding 20 ml of blocking buffer (5% non-fat dry milk, 0.1% Tween-20 in TBS). Incubate for 2 h at room temperature or overnight at 4¡ãC with agitation) •Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) •At what size are the bands migrating? Could they be degradation products of your target? •Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) I have included a picture of secondary only, along with a curve of my samples and control. The bands all look the same as the primary antibody. We do not have milk in this lab for blocking or washing (our gelatin has always worked-ingredients are listed on the ppt slide). Also, secondary reaction would be way to high if it was done more than 1 hour, I am already using a 1:15,000 (a very small amount of secondary antibody) 8. Optimization attempts •How many times have you tried the Western? •Do you obtain the same results every time e.g. are background bands always in the same place? •What steps have you altered? I have tried this primary antibody 6-10 times. It pretty much always looks this bad. I have altered times for incubation, secondary antibody, different combos of primary and secondary, different "positive" controls, increasing the primary conc. 9. Did you apply positive and negative controls along with the samples? Please specify. the only positive control I am aware of that is supposed to work, is the human kidney lysates (ab7920). As far as a neg. control, I don't have any purified protein or anything else on the blots.

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Answer

Thank you for sending the details of your protocol and the picture of your blot. I really think that the background bands are due to the secondary. What appears to be happening is that the secondary is binding additional proteins and reacting with the blocking agent. You need to try to get rid of all the bands in the secondary only control. To do this, I suggest that you change the blocking agent. Try 5% non-fat dried milk with Tween 20 and also try 3% BSA. You may also need to try a lower concentration of the secondary and also, I suggest that you increase your number of washes and make sure that there is Tween 20 in your washes. The gelatin that you use for the blocking and washes may very well be unsuitable with this antibody. Let me know how these suggestions work out for you.

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