Anti-pan-AKT antibody (ab8805)

Thanks for review this technical inquiry and the questions.  Please find below our answers to the questions that you asked, hope it to be of some assistant. Please feel free let me know if there is further question. Thanks a lot.   1  The samples in each lane were the same 2  The proteins were from the same animal-rat 3  The concentration of protein was the same--50ug 4  I don't have whole gel imagines for any of them, we tried to save the antibodies. 5.  Our PVDF membranes had already been prewetted in methanol before use.  

ANSWER:

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimize the results for these product. I would also appreciate if you can confirm some further details:

As each lane was running the same sample we should expect to see a uniform pattern for each. However in the image provided this is definitly not true. This issue is most commonly found with a bad gel. Sometimes they may go bad if stored incorrectly or for too long but most often the cause is that is is run at too high a voltage or with running buffers that have gone bad. It also seems that an 8% gel may be a less than ideal choice for proteins of this size. I recommend 12% SDS-PAGE for these.

I would suggest using new or commercial gels of 12%, running with fresh running buffers at a lower voltage. I would also suggest using BSA 3% in PBS + 0.1% Tween-20 as a blocking buffer for ab8805 and ab66127 and prehaps a casein based blocking buffer such as ab64224http://www.abcam.com/Protein-Block-ab64226.htmlfor use with the phospho-S473 AKT1 antibody (ab66138).

Should the suggestions not improve the results, please do let me know.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

Dear Abcam Technical,   We tested these 3 antibodies several times under same condition, observed, the dilution factor was lower than optimal. The bands were not displayed clear neither.  We would like to understand from perhaps if you have any advice.   Thanks a lot

ANSWER:

Thank you for contacting us.

I am sorry to hear you have been experiencing problems with one of our antibodies. The quality of our products is important to us and I would like to reassure you that we investigate all customer complaints. I have had the opportunity to review the information and images that you have provided and would like to ask a few questions and offer some suggestions that may help.

These products are covered by our Abpromise guarantee.If we cannot remedy this issue and this is a product that you have purchased within the last six months, we will replace or refund it under our Abpromise guarantee, as you are using it according to specifications listed on our datasheet.

In regards each image, were the samples in each lane the same? Were they different animals, protein concentration? These blots in images for ab8805 and ab66138 appear to have not run through and the gel in the image for ab66127 look like it may have deformed during running. Do you happen to have whole gel images for any of these?

The quality of western blots using PVDF membranes will be greatly increased by pre-wetting the membrane in methanol.If this is not something that you have already dome I would suggest doing this. PVDF is hydrophobic and therefore should be prewet in methanol before it is used. Wet the membrane in methanol for 15 seconds. Membrane should uniformly change from opaque to semi-transparent. Carefully place the membrane in ultrapure water and soak for 2 minutes. Then carefully place the membrane in transfer buffer and let equilibrate for at least 5 minutes. You should get a cleaner signal with less antibody when performing this.

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.

Customer requested the information regarding manual quantification of antibody signal in IHC-P.

ANSWER:

Thank you for contacting us.

I would say that this sort of experiment is very-very tricky. We unfortunately do not have any protocol available at the moment for manually IHC staining quantification with antibodies. I would suggest ploughing through the publications and finding if there are scientists who are using similar kind of method.

http://www.histochem.org/archives/photoshop.pdf

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1810239/

http://www.ncbi.nlm.nih.gov/pubmed/21370029

Have a look at these articles and check which one could help you to set up the experiment.

I hope this will help.

Hi, Have you the IgG concentration of this antibody? Is there a peptide block available? Have you noticed lot to lot variability with the antibody?

ANSWER:

Thank you for your enquiry. The total protein concentration is 85 mg/ml (by Refractometry) but we do not know the IgG concentration (the purity of the antibody is whole antiserum). The blocking peptide is available and is catalog # ab9041. There have not been any reports of lot to lot variability with this antibody and please note that our policy at Abcam is that if an antibody does not work as specified on the datasheet, we will offer a replacement or reimbursement.

Please contact us again if you have any additional questions and happy new year!

Customer is interested in using these antibodies in IHC-P. Applications per vial? Does it digestion? Does it need blocking? What controls should be used? Can the controls be obtained from us? Can the digestion/blocking agents be obtained from us?

ANSWER:

Thank you very much for your enquiry. Thus far, I was able to obtain the following information regarding the use of these antibodies in IHC - formalin fixed paraffing embedded sections.

Ab17048 - I would suggest titrating the antibody 1:20 - 1:200 and optimizing based upon results. As with all of these antibodies, the number of applications is going to be dependent upon the dilution used. I would recommend starting with the heat mediated pressure cooker method for antigen retrieval (we have a protocol for this located in the protocols tab on the online datasheet). For a positive control, uPA and uPAR are known to be overexpressed in mesenchymal and epithelial tumor cells. For more details regarding the use of this antibody in IHC, please refer to the following article:

Luther T et al. Epitope-mapped monoclonal antibodies as tools for functional and morphological analyses of the human urokinase receptor in tumor tissue. Am J Pathol 150:1231-44 (1997). PubMed: 9094980

Ab6152 - It is recommended to titrate the antibody 1/10 to 1/100. This antibody requires antigen retrieval using heat treatment prior to staining of paraffin sections. Sodium citrate buffer pH 6.0 is recommended for this purpose. Small intestine or colon tissue is recommended as a positive control for this antibody.

Ab8805 - It is recommended to use this antibody at 1/1000 -1/1500. We have an specific IHC protocol for this antibody, the link is on the online datasheet. There is also a nice IHC image with ab8805 on the online datasheet, normal colon tissue and tumour tissue are compared.

Ab7970 - Our source for this antibody used the antibody in IHC under the following conditions:

The tissue sections were (briefly) pressure cooked for antigen retrieval in citrate buffer pH 6, for 1 minute. The ab7970 was diluted 1:400-1:2000. The primary antibodies were localised using the DAKO ChemMate kit (K5001). Tissues used were human DLBCL and a human inflammatory skin biopsy.

Ab9498 - It is recommended yo use the antibody at 1/10 - 1/25 for 30 - 60mins at RT (ABC method). For formalin fixed paraffin embedded sections, perform enzymatic antigen retrieval using pepsin before commencing with the IHC staining protocol. Tonsil tissue is recommended as a positive control (there is also an image showing this on the online datasheet).

Please note that we have a very nice IHC-P protocol located on our website. it can be accessed by clicking on the protocols tab located on all of our online datasheets. You should do a blocking step with all of these abs; we recommend blocking in 10% normal serum with 1% BSA in TBS for 2 hours at room temperature

I'm still waiting to obtain information regarding ab4802 and ab14195 and I will contact you again once I have received it. Have a great weekend!