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Thank you very much for your response.
I'll be happy to receive the ab96895 secondary Antibody as an alternative to the one currently used to the adress indicated below:
When do you think this could arrive to the school?
I appreciate the information provided regarding with the new set of Ab's that I'm planning to acquire and their compatibility with the conjugation link. I'll be looking forward to your response once the team go back to you.
In answer to your questions:
S phase: I've successfully identified the S phase cells by intercalating an analogue of thymidine within the nascent DNA, for doing so, I've used a kit for cell proliferation from the Life Technologies Company and I detect the newly synthesized DNA by means of fluorescent microscopy. This process is very straightforward, I just need a highly proliferative cell culture and to prepare a solution for it to react with and that's pretty much it!. At the beginning of the experimental design of my PhD I decided to label only G1 and S phase cells and therefore to determine G2 and M phase cells by exclusion; however, due to the lack of specificity in my ICC system I will definitely need to label G2 cells to be sure to be sure which is the developmental state of each single cell within my slide.
Please notice that I haven't run the simultaneous labelling of cells (G1 and S) within the same slide so there is no chance for the S phase labelling reagents to alter the results I've got for the G1 labelling procedure. I decided first to optimize each ICC individually and then combine them all together.
Regarding with the progression of the experiments, I have stained 2 more cell lines onto the growth surface of the plates (LNcap and A-498, prostate and renal cell lines respectively) with no satisfactory results yet. (Unfortunately I haven't got any HeLa specimen so far as my collegues work with GU cell lines mainly).
Thank you in advance,
Looking forwar to hearing from you,
Asked on Apr 11 2012
Thank you for confirming that information. I have arranged for thenew secondary antibody (ab96895) to be sent out to you directly (to the address specified in your last email). The new order number is xxxx(FOCRxxxxx). This should reach you later on today or tomorrow.
I also now have some more input from the lab regarding the compatibility of the anti-Greminin antibody (ab104306)with the Rhodamine conjugation kit. The lab have said thatthey recommend using antibody concentrations of 1-4 mg/mL, asthis generally give the optimal conjugation results. With the antibody being at 0.36 mg/ml, ifused withtheRhodamine Kit, we would expect to not obtain satisfactoryresults as the reactive chemicals would be too dilute.
We would therefore recommendthat the antibody be concentrated prior to carrying out the conjugation procedure. This can be done using the antibody concentration kit, https://www.abcam.com/antibody-concentration-kit-3-columns-ab102778.html.It is worth having a look at the FAQs relating to the conjugation kits we offer which can be accessed from the link below:
The antibodies ab104306 and ab81032 would both be compatible with the kit but if considering using any other antibodies with the kit it is important to check that no substances that are incompatible with the kit are present in the solution (Question 5).
I hope this information has been of help. I look forward to hearing how you get on with the newsecondaryantibody. I'll keep my fingers crossed that the experiment works!
Answered on Apr 11 2012