Anti-Rad17 (phospho S645) antibody (ab3620)

Is it possible for you to send me the ab63442?
Thanks.

ANSWER:

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number 1048217 (ab63442).

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

Thanks for your reply.
I repeated the experiment with a positive and a negative control ordered from another company, and the outcome was not improved.
So I'm wondering if you have any other antibodies for the same target recommended?
Thanks!

ANSWER:

I am sorry to hear that the results does not improve. I can offer you another antibody we have following antibodies against the same target

ab63442; http://www.abcam.com/Rad17-phospho-S645-antibody-ab63442.html

ab115876; http://www.abcam.com/Rad17-phospho-S645-antibody-ab115876.html

Please check ab115876 is a mouse polyclonal antibody, you would need anti mouse secondary if you opt for this.

Let me know which ab you would like to try.

Thanks for your kind reply.
I used human glioma cell line U251 in my study, and I have seen paper reported glioma cells have this pRad activity.
I stripped and reblotted with Actin and got pretty good signal, which i guess means the transfer was good.
I think what you said means using BSA for blocking can only reduce the background noise.
Since I didn't even get any specific binding, I guess try again with BSA may not have signficant effect for my experiment.

ANSWER:

Well that's true. BSA reduces the background only, it does not have any effect on ab specificity.

Anyway to proceed further in this case, I would like to ask if you are happy in trying a different lot or a different antibody. Alternatively I can also provide you full refund or credit note, which you can use for future purchases. Let me know the option you would like to go for.

Many thanks for your cooperation in this case, I highly appreciate that.

Thanks for your reply.
The samples that I used were whole cell lysates from cells 3 hours, 24 hours, and 48 hours post treated with 20 Gy of radiation, as well as cells without radiation as negative control.
So I think the signal should be strong.
By the way, what BSA solution do you suggest as blocking reagent? Do I need to use BSA solution for both primary and secondary antibodies?
Thanks!

ANSWER:

Thank you very much for your email.

We use 5% BSA (Sigma) in TBST for blocking and for diluting secondary and primaries. Milk in not recommended for phospho specific antibodies because milk contains casein which is a phosphoprotein; This is why it causes high background because the phospho-specific antibody detects the casein present in the milk. Please be advised that BSA will only reduce the background i.e. membrane dirtiness, it will not effect the antibody specificity. So if the membrane shows high background then I would certainly recommend trying BSA.

The other important point is, whether the cells naturally express the protein or not. I am sorry the info of cell line wasn't included in your email so I can't be sure about it. I would however recommend using Hela lysates as positive control. Are you sing human cell line?
Actin or GAPDH can be used as loading control. Have you tried any of these?

I hope these suggestions will help to improve your results. If the results do not improve please do not hesitate to contact me.

It will take approximately 5 minutes to complete the questionnaire. Please fill-in all fields so that we can better assist you.

1) Abcam product code ab3620

2) Abcam order reference number or product batch number GR686-4

3) Description of the problem

No signal

4) Sample preparation:

Type of sample (whole cell lysates, fraction, recombinant protein…) whole cell lysates

Lysis buffer RIPA buffer

Protease inhibitors: Protease inhibitor cocktail

Phosphatase inhibitors Phosphatase inhibitor cocktail

Reducing agent SDS

Boiling for ≥5 min? yes, 10 min

Protein loaded ug/lane or cells/lane 51 ug

Positive control n/a

Negative control n/a

5) Percentage of gel 4-20% gradient gel

Type of membrane PVDF

Protein transfer verified ponceau reagent

Blocking agent and concentration 5% milk in TBST

Blocking time 1 hour

Blocking temperature room temperature

6) Primary antibody (If more than one was used, describe in “additional notes”) :

Concentration or dilution 1/500

Diluent buffer 5% milk in TBST

Incubation time overnight

Incubation temperature: 4 degree


7) Secondary antibody:

Species:Donkey

Reacts against: rabbit

Concentration or dilution 1/5000

Diluent buffer 5% milk in TBST

Incubation time 1 hour

Incubation temperature: room temperature

Fluorochrome or enzyme conjugate:HRP

8) Washing after primary and secondary antibodies:

Buffer TBST

Number of washes 3 times, 5 minutes each

9) Detection method Chemiluminascence

10) How many times have you run this staining? twice

Do you obtain the same results every time? yes

What steps have you altered to try and optimize the use of this antibody? no

ANSWER:

Thank you for your enquiry regarding ab3620 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.

The protocol looks fine to me however I would like to make few comments/suggestions that might help to improve your results and that you could consider trying:

ab3620 is a phospho specific antibody. We used 10Gy IR treated Hela cells to test the specificity of this antibody. I would recommend treating your sample cells there by inducing the phosphorylatuion of amino acid chemically or by radiations.

Milk is not recommended as blocking reagent with phospho specific antibodies. Please use BSA.

Please try both of these suggestions and let us know the results; we will be happy to help further.