Anti-Human intestinal alkaline phosphatase antibody (ab7322)

Phone call: Customer kindly expressed concerns about the specificity of antibody ab7322. The antibody is detecting a band at 60 kDa in T cell line lysates which is supposed to be negative for AP. - T cell line was the only cell line used as negative control. - Antibody detected a band of right size at 60kDa in intestinal cell line. 

ANSWER:

Thank you for contacting us.

The antibody hasn't been tested for its cross reactivity with other types of APases. The sequence homology between these APases is quite significant so it may be possible that this antibody will cross react. Click to check the homology

http://www.uniprot.org/blast/uniprot/20111103306OTC8JBT

T-cells doe shave alkaline phosphatases in these. There are publications that guide the presence of APases in T cell also. Please find the link below

http://cancerres.aacrjournals.org/content/43/11/5416.full.pdf

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1904623/pdf/cei0108-0019.pdf

I probably suggest using other cell line as a negative control.

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

Phone call: Customer kindly expressed concerns about the specificity of antibody ab7322. The antibody is detecting a band at 60 kDa in T cell line lysates which is supposed to be negative for AP. - T cell line was the only cell line used as negative control. - Antibody detected a band of right size at 60kDa in intestinal cell line. 

ANSWER:

Thank you for contacting us.

I can confirm that I have logged your inquiry in CRM system and now checking the details you have asked. I will get back to you with more information soon.

Many thanks for having patience. Please do not hesitate to contact us if you have any additional question. 

Dear Colleague,

I hope you can help me with this enquiry.

I figured SEAP = secreted alkaline phosphatase. ab7322 is directed against alkaline Phosphatase [human intestine]. Was the secreted or the intracellular alkaline phosphatase used as the immuogen. Do you know if ab7322 recognizes both forms - has it been tested?

ANSWER:

Dear Dr. Ancellin,

Thank you for your enquiry and patience.

In regards to ab7322 (human intestine alkaline phosphatase Ab)in Western blotting (reducing SDS-PAGE), this antibody detects a 60,000 dalton protein from cellular extracts.

ab7322 has not been specifically tested against the two forms of the human intestine alkaline phosphatase, secreted and intracellular.

The immunogen used in producing ab7322 is the full protein - Alkaline Phosphatase [human intestine]. If the homology between the two forms are identical/high, then there is a good chance that ab7322 will recognize the secreted form.

I hope this information helps and please inform me of your experimental results.

Well I have a question on ab7322. What is the concentration on the western blot, and the suggested dilutions for ab7322?

ANSWER:

Thank you for your enquiry. The starting dilution that was employed in order to detect 10ug total protein derived lkaline phosphatase (human intestine) was 1:10000. The dilution factor used may then need to be optimised, depending upon the results gained during this experiment.

I would like to know if this ab has some known cross reactivity against placental human alkaline phosphatase. I would like to use it for stainig a transmembrane construct. Unfortuantely i´m not sure which alkaline phosphatase is included in this transmembrane construct.

ANSWER:

We have no information as to whether this antibody cross-reacts with placental human alkaline phosphatase. It was raised against human intestinal alkaline phosphatase, it may cross react with placental alkaline phosphatase depending on the homology.