Dear Jon,

Thanks for your reply.

Yes, I have a wash step after my protein-aptamer complex is passed through. ( I do it once. Is there a need for more washes???)

And does the Incubation time and temperature make a difference with the data?

short or long incubation     AND

-20C or RT ?

In your experience which is the best for reproducible results?

And I don't have access to the protocol link you have attached. Can you please send me a copy of it If you have access. I would really appreciate it.

Static on the surface of tube could be a problem form some scintillation counter. Try to put the tubes in ice for few seconds just before counting and see if it changes your results.

I do not work with Protein´s only with sequencing of eDNA. But with my eDNA samples I used a DNA repair mix after extraction. It helped me a lot for my downstream reactions. Suggestable that because of the repairing the proteins and enzymes could better bound on the DNA. Maybe that helps you too.

What steps do you take to purify the eDNA.  After labeling it is common for the majority of 32P to be unincorporated.  In order to measure the specific activity of the eDNA (in cpm/ul, for example), you first need to get rid of unincorporated label using a size exclusion column, or the like.  You can use products like these (see links below).

http://www.gelifesciences.com/webapp/wcs/stores/servlet/ProductDisplay?categoryId=11520&catalogId=10101&productId=21009&storeId=11787&langId=-1

http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-us/products/AlternativeProductStructure_17513/27532501

Dear Chandan

I recommend you make them filter binding assays in membranes of nitrocelulose in a dot-blot system. This method allows you to use higher volumes of incubation and concentrate each sample. Uses samples with 10 ug of target protein as maximun. Perform the binding assays in solution and then filter it in the membrane from nitrocellulose, rinse three times with PBS. The membrane can be exposed it wet with folex paper and you can quantify the retained aptamer with autorradiografic films and later retrieve ligands concentrates in the dot. You can retrive them by heating to 95 ° C for 5 min at H2O miliQ and verify their presence and quantity by Cherencoff and later, the use aliquots for more amplifications (Radrizzani el. al, 1999 and Moncalero et. Al, 2011). Good luck,

Martin

Dear Jon,

I am confused a little.

Firstly, about the incubation temperature. I was actually referring to incubation temperature after the assay, for development on the film. Once we are through the filter binding and vacuum suction, we need to keep the plate on the film for getting the exposure data right? I was referring to this step.

I usually incubate the film for O/N exposure and at -20C.

Please reply to the above in terms of any variations with temperature and duration of incubation of the film, would that affect my data?

And may be i can try with 3X washes, i usually do the washing simultaneously (first wash) after i add my prot-eDNA mix to the plate. And after all the samples are added, I will do a wash once more to clear out the non specific binders adhering to the plate as such.

Dear Frederic,

Most of the times, it so happens that I take out my eDNA out of -20C and in a span of 10-15min I take the counts. Anyways, I can try out your suggestion the next time.

Thank you so much.

Dear Paromita,

I do not have the access to the protocol. Please send me a copy if you have access.

Ya sure, I will have 3X washes in my next experiments. And ya, I am aware of the half life. So, we usually check the cpm counts before the experiment and normalize the labelled DNA content to 2000cpm/well.

Is 2000cpm/well labelled content enough? as per your experience in a 96W format?

Dear Babett,

May be i can try that too. Do you have any references for your suggestion? So, that I can go through it once before I could try them.

Thank you.

Dear Dan,

We use the microspin G-25 columns.

But I have found out that, even after this step often we tend to get the unlabelled portion in a major chunk. How to avoid that? (We spin at 800RCF 2min)

I also tried to concentrate my eDNA by extracting them in a lower volume, this seems to enrich the labelled portion to quite a bit.

But always going back to the bead, extracting the bound eDNA again in lower volume is additional work. Is there anyway that i can get out of this problem.

Thanks.

Dear Martin,

Thanks for your suggestions. Will go through the papers you have mentioned and think about your valuable inputs.

Dear Jon,

I think you are mentioning about recovery of the bound aptamers from the membrane?