Anti-Geminin antibody (ab104306)

Thank you for contacting us.

The following protocol was used with ab104306 for ICC/IF:

Preparation of slides:

1. Grow cultured cells on sterile glass cover slips at 37°C overnight.

2. Wash sample with PBS twice.

3. Fix cells for 15 minutes with 2 mL of 4% paraformaldehyde solution (pH 7.4 with NaOH in PBS).

Step-by-step procedure:

1. Permeabilize cells by incubating for 15 minutes on ice with 2 mL of 0.1% Triton X-100 in PBS.

2. Wash cells 3 times with PBS.

3. Incubate cells for 1 hour with normal goat blocking serum (1:20 in PBS).

4. Introduce primary antibodies (in appropriate dilutions) to the sample.

5. Incubate for 4 hours at room temperature or at 4°C overnight.

6. Wash with PBS for 5 minutes. Repeat 5 times.

7. Incubate cover slips in fluorescein-conjugated secondary antibodies in 5% normal blocking serum in PBS in a dark humidity chamber at 4°C for 1 hour.

(Perform all subsequent washes under dim and ambient light source.)

8. Wash sample thoroughly with PBS. Each wash lasting 5 minutes. Repeat 6 times.

9. Counter stain sample with DAPI at room temperature for 30 minutes.

10. Wash sample with PBS. Each wash lasting for 2 minutes. Repeat 3 times.

11. Mount sample by inverting them onto mounting medium on glass slides.

12. Store slides in dark at 4°C.


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

Thank you for your reply.

As I mentioned in my previous email, we do not have sample sizes of our products. Instead we guarantee them, under our Abpromise to work as stated on the datasheet. If for whatever reason they fail to work, then we will replace or refund the antibody for you.

For ab104306 we guarantee to work on human tissue in the following applications: ELISA, IHC-P, ICC/IF, WB. If you wish to try the antibody on a different species, then you can take advantage of our 100% Abreview testing Discount program:

https://www.abcam.com/index.html?pageconfig=resource&rid=11998&viapagetrap=collaborationdiscount



If you would like to take advantage of the testing discount program or if there is anything else I can help you with, please let me know.

Thank you for contacting Abccam. Because we carry over 90,000 products, it isn't feasible for us to keep small sample sizes of our products.

We are happy to reassure our customers that all of our products are covered by our Abpromise, which guarantees that the product will work in the applications and species specified on the datasheet, or we will offer a replacement, credit, or refund within 6 months of purchase.

If the product is to be used in an untested species or application, you may be eligible for our testing discount program if the antibody has not yet been purchased. Please contact our Scientific Support team by replying to this email prior to purchase for more information.

Otherwise, we like to encourage all of our customers to submit an Abreview via the online product datasheet. We always appreciate customer feedback, whether positive or negative, and we make all product information available to researchers. Plus, each Abreview earns Abpoints that can be used for discounts on future purchases or rewards such as Amazon.com gift certificates.

To find out more about our Abreview system, please see the following link:

https://www.abcam.com/abreviews

I hope this information is helpful. Please do not hesitate to contact us again with any other questions.

I'm glad to hear it. If you do have any further questions please do not hesitate to contact me again.

Until then, I wish you all the best with your experiments.

I am sorry that the protocol details were not explained clearly enough. I will try to explain as best I can, if it does not make sense please let me know and I'll try to rephrase.

You would take your antibody (concentration of 0.36 mg/mL), you are hoping to achieve a concentration of at least 1 mg/mL to use it with theconjugation kit. In order to do this, you could concentrate all the antibody you will be provided with:

100 ug of antibody at 0.36 mg/mL, gives you 277.7 uL in total. This therefore needs to be concentrated by about a factor of 3 to get the desired concentration. You would therefore load the antibody into the spin cartridge. Spin (start with no more than 1 minute at no more than 15,000g), this will give you an idea of how quickly the solution will be removed. Continue to spin (checking after every minute or less or centrifugation) until you have removed ˜185 uL of solution. You should therefor be left with ˜92 uL of antibody at ˜ 1mg/mL.

If you would like to concentrate only a portion of the antibody, the same process can be followed but reducing the volume added and the volume discarded. Be very careful however to not let the antibody come to complete dryness (try not to reduce the antibody volume to below50-100 uL).

I hope this has helped to clarify the procedure. If you have any further questions, please do not hesitate to contact me again.

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:

https://www.abcam.com/index.html?pageconfig=resource&rid=13156

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!

I'm sorry for the delay in getting back to you, we have been closed over the Easter holidays.

I have had a look at the secondary antibodies you have suggested as trying as an alternative to the one currently used (ab60314). All seem fine for the application you hope to use. If you would like I can send this out to you free of charge. Unless you have a preference for a different one, I'd send out ab96895.You mentioned that you were in Ireland? Would you still like for this replacement secondary antibody to be sent to the address I have for you:

xxxx


I am not sure how the antibody concentration of anti-Geminin antibody ab104306 would affect the use in conjunction with the conjugation kit (ab102915). If will forward this query to the source of this kit and will let you know as soon as I have a response. As you will be conjugating directly, I would suggest that using an isotype control would be quite important asyou are not able to do a "no primary" control experiment to ascertain the non-specificity observed.Mouse monoclonalab81032 should be suitableand would becompatiblewiththe conjugationkit.

Can I ask, how are you going to detect the S phase (with Alexa Fluor 647), are you using another antibody? In which case, what antibodies do you intend to use?

How have your subsequent experimentsprogressed? Have you used the HeLa cellswith adherent slides?

I hope this information has been of help.I look forward toreceivingyour response.