Thank you so much, Keith!
Yaroslava
From: technical@abcam.com [mailto:technical@abcam.com]
Sent: Monday, April 30, 2012 7:13 PM
To: Ruzankina, Yaroslava (NIH/NCI) [E]
Subject: Reply from Abcam to your enquiry regarding ab85898 [CCE3727685]
http://www.abcam.com/index.html?utm_campaign=CRM&utm_source=Abcam.CRM&utm_medium=Email&utm_term=Header
Dear Dr Ruzankina
Thank you for contacting us. I have tried to answer your questions below and have attached our IHC-P protocol for your reference.
1) When you do antigen retrieval for ab85898, for how long do you boil in citric buffer? We recommend boiling in a pressure cooker 3 minutes. However, three minutes is only suggested as a starting point antigen retrieval time. Less than 3 minutes may leave the antigens under-retrieved, leading to weak staining. More than 3 minutes may leave them over retrieved, leading to non-specific background staining and also increasing the chances of sections dissociating from the slides. A control experiment is recommended beforehand, where slides of the same tissue section are retrieved for 1, 2, 3, 4 and 5 minutes before being immunohistochemically stained to evaluate optimum antigen retrieval time for the particular antibody being used.
The use of a domestic microwave is inadvisable. Hot and cold spots are common, leading to uneven antigen retrieval. Antigen retrieval times are usually longer,˜10 minutes due to the absence of a pressurized environment, nearly always leading to section dissociation. When using this method, it is possible for the buffer to boil over, and a large amount of the retrieval buffer will evaporate. Be sure to watch the buffer level of the slide vessel, and add more buffer if necessary. Do not allow the slides to dry out.
2) When you incubate with ab85898 antibodies, what solution do you use for mouse tissues (IHC-P)? Is it the same solution that you do blocking with (10% serum, 1%BSA in TBS)?
We recommend incubation in TBS with 1% BSA, however many groups do incubate in that same solution without problem.
3) What conditions do you use for ab72130 (anti-Smoothened) – mouse tissues IHC-P? What would be the antigen retrieval, blocking and diluent for primary antibody?
I would recommend the same conditions as above. While the exact concentration of ab72130 will need to be optimized empirically however it does appear that a concentration of 10ug/ml would be a good starting concentration.
4) What conditions do you use for ab7195 (anti-Gli2) for mouse IHC-P?
Anti-Gli2 has been used in several publications in IHC-P. These groups were able to use the same methods which we recommend for the majority of our IHC-P validated products. These publications fixed the tissue with 4% pfa, used Citrate buffer pH6 for antigen retrieval and used dilutions around ˜1:200. These exact conditions required for your sample will need to be optimized in lab.
I have attached our complete IHC protocols with this message. This contains many of the recommendations which we have spoken about as well as other information about IHC which you may find useful.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
Use our products? Submit an Abreview. Earn rewards!
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Help us improve our service.
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Best regards,
Keith
Keith Beadle
Scientific Support Specialist
Abcam Inc.
www.abcam.com
Your original inquiry to Abcam:
Dear Keith,
Could you answer a few more questions.
1) When you do antigen retrieval for ab85898, for how long do you boil in citric buffer?
2) When you incubate with ab85898 antibodies, what solution do you use for mouse tissues (IHC-P)? Is it the same solution that you do blocking with (10% serum, 1%BSA in TBS)?
3) What conditions do you use for ab72130 (anti-Smoothened) – mouse tissues IHC-P? What would be the antigen retrieval, blocking and diluent for primary antibody?
4) What conditions do you use for ab7195 (anti-Gli2) for mouse IHC-P?
Thank you!
Yaroslava
From: technical@abcam.com [mailto:technical@abcam.com]
Sent: Tuesday, April 24, 2012 2:53 PM
To: Ruzankina, Yaroslava (NIH/NCI) [E]
Subject: Reply from Abcam to your enquiry regarding ab85898 [CCE3711661]
Dear Dr Ruzankina
You are very welcome.
Please remember that these products are covered by our Abpromise guarantee. We provide scientific support, replacement or refund should this product not perform as indicated on the datasheet. More information on our Abpromise may be found at the following link:
http://www.abcam.com/index.html?pageconfig=abpromise
I would like to encourage you to take advantage of our Abreview program. It takes just a few minutes to leave a review and you can collect Abpoints which you may redeem for Abcam products or Amazon gift cards while at the same time sharing information about the product with your colleagues worldwide. More information may be at:
http://www.abcam.com/index.html?pageconfig=resource&rid=10332
I hope that this information is helpful. Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.
Help us improve our service.
http://www.abcam.com/index.html?pageConfig=technicalSurvey&intCCEID=3711661
Best regards,
Keith
Keith Beadle
Scientific Support Specialist
Abcam Inc.
www.abcam.com
Your original inquiry to Abcam:
Dear Keith Beadle,
Thank you so much for your prompt response! We were trying to figure out if we should order these antibodies and now after your reply, we will order them.
Thank you!
Yaroslava
From: technical@abcam.com [mailto:technical@abcam.com]
Sent: Tuesday, April 24, 2012 2:29 PM
To: Ruzankina, Yaroslava (NIH/NCI) [E]
Subject: Reply from Abcam to your enquiry regarding ab85898 [CCE3711528]
Dear Dr Ruzankina
Thank you for contacting us.
This product has been tested and guaranteed under our Abpromise to work in IHC-P on mouse tissues. We recommend using heat induced antigen retrieval using a Sodium Citrate buffer pH 6.0. We do offer the 10x Heat Mediated Antigen Retrieval Solution pH 6.0 as product number ab973 (http://www.abcam.com/10x-Heat-Mediated-Antigen-Retrieval-Solution-pH-6-0-ab973.html).
The recommended dilution of ab85898 in IHC-P is 1:300- 1:2000 however because differences in protocol, tissue or antibody lot, the optimal dilution factor in your experiments will have to be determined in lab.
We also recommend blocking with a 10% normal serum with 1% BSA in TBS for 2 hours at room temperature.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
Use our products? Submit an Abreview. Earn rewards!
http://www.abcam.com/abreviews
Help us improve our service.
http://www.abcam.com/index.html?pageConfig=technicalSurvey&intCCEID=3711528
Best regards,
Keith
Keith Beadle
Scientific Support Specialist
Abcam Inc.
www.abcam.com
Your original inquiry to Abcam:
Dear Representative,
I am writing to inquire about anti-Pim1 antibody (ab85898).
Have you tried it on mouse tissues (IHC-P)? If yes, what conditions would you recommend (antigen retrieval, dilution etc.)?
Thank you.
Yaroslava Ruzankina, PhD
National Cancer Institute - Frederick
MCGP/CPM
Bldg 560, Rm 32-24
Ft. Detrick, MD 21702
Phone: 301-228-4890
Fax: 301-846-1290
Abcam Customer Services and Scientific Support Team
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[CCE3711528]
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Abcam Customer Services and Scientific Support Team
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[CCE3711661]
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[CCE3727685]
Discover more at abcam.com |
ANSWER: |
|
Thank you for contacting us. I have tried to answer your questions below and have attached our IHC-P protocol for your reference.
1) When you do antigen retrieval for ab85898, for how long do you boil in citric buffer?We recommend boiling in a pressure cooker 3 minutes. However, three minutes is only suggested as a starting point antigen retrieval time. Less than 3 minutes may leave the antigens under-retrieved, leading to weak staining. More than 3 minutes may leave them over retrieved, leading to non-specific background staining and also increasing the chances of sections dissociating from the slides. A control experiment is recommended beforehand, where slides of the same tissue section are retrieved for 1, 2, 3, 4 and 5 minutes before being immunohistochemically stained to evaluate optimum antigen retrieval time for the particular antibody being used.
The use of a domestic microwave is inadvisable. Hot and cold spots are common, leading to uneven antigen retrieval. Antigen retrieval times are usually longer,˜10 minutes due to the absence of a pressurized environment, nearly always leading to section dissociation. When using this method, it is possible for the buffer to boil over, and a large amount of the retrieval buffer will evaporate. Be sure to watch the buffer level of the slide vessel, and add more buffer if necessary. Do not allow the slides to dry out.
2)When you incubate with ab85898 antibodies, what solution do you use for mouse tissues (IHC-P)? Is it the same solution that you do blocking with (10% serum, 1%BSA in TBS)?
We recommend incubation inTBS with 1% BSA, however many groups do incubate in that same solution without problem.
3)What conditions do you use for ab72130 (anti-Smoothened) – mouse tissues IHC-P? What would be the antigen retrieval, blocking and diluent for primary antibody?
I would recommend the same conditions as above. While the exact concentration of ab72130 will need to be optimized empirically however it does appear that a concentration of 10ug/ml would be a good starting concentration.
4)What conditions do you use for ab7195 (anti-Gli2) for mouse IHC-P?
Anti-Gli2 has been used in several publications in IHC-P. These groups were able to use the same methods which we recommend for the majority of our IHC-P validated products. These publications fixed the tissue with 4% pfa, used Citrate buffer pH6 for antigen retrieval and used dilutions around ˜1:200. These exact conditions required for your sample will need to be optimized in lab.
I have attached our complete IHC protocols with this message. This contains many of the recommendations which we have spoken about as well as other information about IHC which you may find useful.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
Use our products? Submit an Abreview. Earn rewards!
http://www.abcam.com/abreviews |
Hi
The Gli2 protein actually comes in two forms; the 165 kDA (actually larger due to post translational mods) and 75kDA cleaved form which acts as a weak pathway repressor (Gli3 is the main repressor). There is a paper by Baolin Wang (Pan et al, 2006) that describes this very well and they are the lab that gave me the other antibody. If you look closely there are actually 2 bands around 165kDA which are very faint although these are hard to see, there is another higher up as well. I am not too concerned about how weak this is as ultimately I will be using Biotin oligo pull downs to concentrate Gli proteins. I have now seen enough from this antibody to progress it to the next stage. The Abcam antibody has given me absolutely nothing so I am concerned about using it in the next stage which is more time consuming and expensive. I simply just want to see that the antibody is detecting something, even if it is not exactly what I expect. I should also mention that the Abcam antibody will not detect a 75kDa band as it is raised against epitopes on the proteins C terminal and this section is the part that actually gets cleaved. This is why I want to use both these antibodies as they are raised against opposite ends of the protein. However first I must see in this very crude way that the antibody works and is not a dud.
I have already used PVDF which is much better but also got nothing with your antibody, have not tried it with the other antibody. I will use PVDF in future but as I have a lot of nitrocellulose and just want to see some crude preliminary evidence of detection at this stage, I am not too concerned about the background and other problems nitrocellulose is giving me.
The ladder brightness is due to the increased brightness i have purposefully contrived on these images to highlight some of the weaker bands. I will be increasing the protein I use (50ug is not possible, perhaps 25ug) and purifying/enriching. I fully expect not having to make the images so bright in the future. I will nevertheless reduce the ladder as it is a good idea anyway.
your suggestion that I use higher antibody concentrations is a good idea and what i want to do next, although I am concerned about the background this will create. However given that I am to use up so much more of your antibody at these preliminary stages and that I have shown enough to at least give reasonable doubt the antibody I have purchased is faulty, can you please send me a fresh lot to proceed with as I do not have exhaustive time and resources to proceed with. I simply will not be able to keep trying different things to prove your antibody works and let’s face it there will always be different things to try. I have shown my controls work and another antibody has worked, this surely must be enough.
Many Thanks, hope you can help |
ANSWER: |
|
Sorry for the delayin getting back to you. We have been closedover the Easter weekendperiod.
Thank you for getting back to me with that information.I can well understand your point of view. My concern is that, as you say, our antibody is supposed to only detect the non-cleaved substrate and would only produce the band at ˜165 kDa. This band is also very weakly detected using the alternative mouse monoclonal antibody which you have. This makes it difficult to ascertain if it is the protein sample which is not strong enough or if it is the antibody which is faulty. This is why I have made the suggestions that I have, in order to potentially improve the results.
As a goodwill gesture I am able to provide you with a newvial of ab7195 to try. Unfortunately, we only have antibody form the same bleed to offer. However, as we have had no reports of similar issues with this lot (or any other lot of this antibody sold in the last year) I thinkif it is a problem of the antibody, it is likely to be isolated to the vial you received. This can unfortunately happen from time to time due to the nature of the product itself. If you would like to receive this new vial please do let me know and I will have this arranged as soon as possible.
If you would prefer to try an alternative antibody, such as anti-Gli2 antibody (ab26056), I can also have this arranged. This antibody is raised against a synthetic peptide taken from the region of 272 - 321 of human Gli2 (but it has been shown to detect the mouse protein).
I look forward to hearing how you would like to proceed. |
