Goat Anti-Rabbit IgG H&L (Alkaline Phosphatase) preadsorbed (ab7091)

Overview

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

    Chicken, Cow, Goat, Guinea pig, Hamster, Horse, Human, Mouse, Rat, Sheep more details
  • Immunogen

    Full length native Rabbit IgG (purified).

  • Conjugation
    Alkaline Phosphatase

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: 8.00
    Preservative: 0.1% Sodium azide
    Constituents: 0.0014% Zinc chloride, 0.0095% Magnesium chloride, 0.79% Tris HCl, 50% Glycerol, 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 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
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab7091 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
ICC/IF 1/200 - 1/1000.
IHC-P Use at an assay dependent dilution.
WB 1/500 - 1/2500.
ELISA 1/8000.
Dot blot Use at an assay dependent dilution.
IHC-Fr Use at an assay dependent dilution.

References

ab7091 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

Question

Attached to the present e-mail is a description of the ELISA we are trying to optimize here in our lab. We are experiencing some problems, but can not figure out where exactly. The products which we get from Abcam seem to work correctly (ab2644 and ab7091) but we were wondering if there was something ion the rest of our technique which interfered with one of them. I hope you can give us some advice on this assay. 1. Which type of ELISA plates have you used? Are they specially treated? The ELISA plates are 96 well flat-bottom Immuno plates, MaxiSorp, non-sterile, From Nalgene Nunc International (#442404) 2. What dilution (concentration) of primary and secondary antibodies have you been using? Ab2644: best results were with 1:1000 in PBS, but we tested ranges from 1:500 to 1:5000 Ab7091: best results were with 1:3000 in PBS, but we tested ranges from 1:1500 to 1:4000 3. I agree it is a concern that the primary antibody (ab2644) may be binding to the plate. Have you tested this by staining a plate with no control pantothenic acid congugate and no blocking agent? What was the result? Did it still bind and give a positive result in all wells? I did test this for some wells on a plate. I did not coat nor block the plate (therefore no pantothenic acid conjugate nor blocking agent) and no standard or sample. I therefore only added 100 uL of 1:1000 primary antibody (ab2644) and then followed the rest of the procedure (200 uL of secondary antibody ab7091 and 200 uL of substrate, with incubation times and washings as mentioned in the procedure). I got positive results in all these wells, although the rest of the tests performed on the same plate did not color for most of them. This is why we suspected that the primary antibody could bind the plate. In addition, in the tests wehere we were trying to get the optimal coating concentration and conditions, we noted that the dilution of the primary antibody was much more related to the intensity of the response than was either the coating or blocking agent concentration, and the pantothenic acid standard concentration. This further emphasized that the more primary antibody we had in the well, the more intense was the response. The hypothesis that this primary antibody binds to the plate would explain these results... 4. Did you wash the plate after adding the Standard pantothenic acid before adding primary antibody? No. The plate was washed to remove the excess coating. The standard pantothenic acid and primary antibody were both added and incubated together for 1.5 hour. The competition for the primary antibody is therefore between the fixed pantothenic acid (from the coating) and the free pantothenic acid (from the standard). We suspected that the length of the incubation could maybe favour binding to the fixed pantothenic acid, and therefore tried to reduce both incubation times (after the primary and the secondary antibodies are added). Results revealed that the responses only diminish in intensity after a shorter incubation, but we did not get better results. 5. At which stage did you add the blocking agents? When the coating used was a pantothenic acid conjugate (Pantothenic-acid-KLH in most cases and some tests with pantothenic-acid-BSA), the plate was incubated overnight, rinsed the next morning, and the blocking agent was then added. The plate was incubated again for 1h at 37 degrees C, and was rinsed. The pantothenic acid standard and the primary antibody were then added, and the plate incubated and all the rest of the method was just as mentioned in the procedure. In the tests where we diluted the pantothenic acid in a blocking agent, we would coat the wells with different concentrations of pantothenic acid in a blocking agent. Example: 50 ug/mL of pantothenic acid in 5% fish gelatine (which is diluted in PBS). The coating was then left in the wells overnight, and the plate was washed in the morning, followed by the addition of pantothenic acid standard and primary antibody... and the rest of the procedure.

Read More
Answer

Thank you once again for your enquiry. I apologise for the long delay in responding to your last message. I have been waiting to receive further information from the laboratory. Antibody ab2644 has been developed for semi-quantitative detection of D.Pantothenic acid. This small molecule must be conjugated to a protein carrier for coating and competition assays. BSA can be used as protein carrier since the antibody has been immuno-purified on this protein during the purification process. The following protocol was used for the characterisation of this antibody: 1. Coating of D.Pantothenic acid-BSA conjugate (10µg/ml, 200µl by well) in maxisorp well plates (Nunc) with a solution of sodium carbonate buffer 0.05M (pH 9,6), during sixteen hours at 4°C. Negative controls are realised with unconjugated BSA at the same concentration and in the same buffer. BSA conjugated D.Pantothenic acid is commercially available. 2. Wash the plates with of a solution of PBS (pH 7,3) containing 1g/l of BSA , 10% of glycerol and 0.5% Tween (one hour at 37°C). 3. Wash three times with PBS containing 0.5% of Tween (PBS Tween). 4. Preabsorbed anti-D.pantothenic acid serum diluted (1/5,000-1/10,000) in PBS Tween containing 1g/l BSA and 10% of glycerol, 200µl by well plate (incubating during 2 hours at 37°C). For affinity and specificity assays (to determine the cross-reactivity ratios), competition scales are made with conjugated pantothenic acid and other competitors. 5. Wash three times with PBS Tween. 6. Add 200µl of conjugated secondary antibody in a solution of PBS Tween containing 1g/l of BSA to each well (incubate one hour at 37°C) . 7. Wash plates three times with PBS Tween. 8. Add 200µl by well plate of a citrate 0,1M/phosphate 0,2M (pH 5) solution containing 0.4% of OPD (Sigma) and 0.03% of hydrogen peroxide for ten minutes in the dark to develop the plates. 9. Reaction stopped by addition of 50µl of 2M HCl. 10. Optical density measured at 492nm. I hope this information is helpful. Should you have any further questions, or experience further difficulties with this procedure, please do not hesitate to contact us again.

Read More

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

Sign up