Hi Nidhi

Your query is little bit confusing to me.....are you really refering to dierctly conjugating the AP enzyme to DNA or are you looking for information on estimating labelling efficiency of non-radioactive labelling protocols (such as DIG based methods) which employs antibody linked AP enzyme for detection ...

bye

Hi Ajai

yes i am using the kit "alkphos direct labelling and detection" from amersham. but i do not really understand the chemisty behind it. the kit uses chemical crosslinking (by formaldehyde) to label the DNA molecule.

radioactive probes have a defined amount of radioactivity integration onto the probes. i want to know the same for enzyme labelled probes. i read some literature, but could not really get anything very informative. please help

Hi Nidhi

I am sure you have gone through product manual of the 'Alkphos direct labeling and detection kit' from Amersham...The chemistry behind is simple as far as detection is concerned as the Alkaline phosphate enzyme is used in most of the non-radiaoactive detection kits form several companies....only the linking of AP is different in this case...it is directly conjugated to the DNA unlike in the kits from other companes...

Regarding the evaluation of efficiency...you can use th approach described in the manuals of other companies such as Roche.....where a serial dilution of sample and a labeled standard DNA is spotted on a nylon membrane and the compare the spot intensities....see the attached document from two companies to check the labeling efficiency of the non-radioactive labelling protocols

Sorry there was some problem in the attachement ...kindly check the 'Roche website'

for DIG labelling kit and estimating efficiency of labeling

bye

Thanks.... I shall go through the manual.