Anti-Rb (phospho S780) antibody [E182] (ab32513)

Customer kindly called to inquire whether this product would recognize hyperphoshorylated Rb.

ANSWER:

Thank you for contacting us.

I have had the opportunity to discuss this with the lab. Unfortunately, we have not tested whether this product would recognize the hyperphosphoylated Rb protein. We cannot guarantee that it will work.

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

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datasheet states IHC is not suitable with this antibody

human tonsil and breast tissue - IHC
sees high background, lot variations (GR12953-1, GR12953-2)
Ab: 1/500, 1/1000, 1/2000 for 30 min
H2O2 block
no protein block included
uses Invision Dual Link for 30min
DAB for 10 min
why is IHC listed as not suitable? any further advice to lot variations

ANSWER:

Thank you for contacting us.

I have heard back from the lab with the following information:

We found negative results on a cancer TMA, but no other details are available.
The concentration varies from lot to lot, but the same batch should produce similar staining.

I apologize that more detailed information was not available from the lab.

As we discussed over the phone, I would suggest to include a blocking step to reduce the background.

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

Attached are my images for IHC. As I mentioned results are variable from run to run.

ANSWER:

Thank you for your patience as I have obtained the following information from the laboratory:


Regarding the antibody being unsuitable for IHC, we found negative staining using a cancer TMA. No additional troubleshooting was done. I would be happy to offer a different lot if you would like to test it. Please let me know what you would prefer.

I apologize for the inconvenience this causes. I look forward to your reply so that I may assist you further.

I saw very nice stainings with the products from your company,as shown
in the following website:
http://www.abcam.com/Rb-phospho-S780-antibody-E182-ab32513.html
About lambda phosphatase, which showed very prominent effects with the
immunostaining. I checked that most information about lambda
phosphatase are western blot. I want to know how was lambda
phosphatase used in your immunostaining assay? Are they used before
fixation or after fixation? Is there any reference about that?

ANSWER:

Thank you for contacting us.

I have received the answers to your questions:

1) Lambda phosphatase is usedafter fixation.
2) Unfortunately, we do not have any product references at this time.

3) The Lambda phosphatase treatment protocol is below:

Materials and Reagents:

LP Buffer
-Combine: 2.5ml 1M TrisHCl, 0.1ml 1M MnCl2, 250ul 1M DTT, and 5mg BSA in 47ml dH2O. Adjust pH to 7.4.

Lambda Phosphatase working solution
-600 units LP in 1ml LP Buffer
“Experiment” tissue/cell slide: LP blocking + IHC/ICC protocol.
“Control” tissue/cell slide: IHC/ICC protocol

Procedure:

1. Adopt tissues/cells to LP buffer:
-Before entering the 10% goat serum blocking step in IHC or ICC, cover both the “experiment” tissue slide & “control” tissue/cell slide in LP Buffer and incubate for 45 minutes
2. After 45 minutes LP blocking:
-Leave “control” tissue/cell slide in LP Buffer for 1 more hour.
-Remove LP Buffer and add LP working solution to the “experiment” tissue slide for 1 hour.
Continue with usual IHC/ICC protocol

Note:
LP buffer should be clear, if the solution appears brown, it is too basic.

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

Concentration of antibody.

ANSWER:

Thank your calling Abcam earlier today. The concentration of ab32513, lot #GR12953-2 is ******* mg/mL. Please letme know if there is anything else I can help you with.