Anti-Angiopoietin 1 antibody (ab8451)

Thank you for contacting us.
I changed the review to editing status. You can now change the Abreview by correcting the details.
Please also confirm that the secondary antibody was polyclonal or monoclonal?
ab8451 is a rabbit antibody so secondary antibody can't rabbit.
Please check these and update the Abreview. Thanks!

Vielen Dank für diese weiteren Informationen.
Es tut mir Leid, dass Sie Probleme mit diesem Antikörper hatten. Wie am Telefon besprochen, habe ich eine kostenlose Ersatzlieferung für Sie in Auftrag gegeben. Sie hat die Referenznummer 123456 und sollte morgen bei Ihnen ankommen.
Ich kann die Blots bezüglich der KO Mäuse gut gebrauchen: Das gibt mir den Rückhalt gegenüber dem Labor nicht nur das betroffene Lot zurück zurufen, sondern dass die Lots in Produktion noch einmal genauer angeschaut werden!
Ich wünsche Ihnen viel Erfolg mit Ihren Projekten. Bitte melden Sie sich wieder bei uns, falls es weitere Fragen oder Probleme gibt.

Es tut mir Leid, dass Sie Probleme mit diesen Antikörpern hatten. Wie am Telefon besprochen habe ich eine kostenlose Ersatzlieferung für Sie in Auftrag gegeben. Sie hat die Referenznummer 123456 und sollte allerdings erst am 5 September bei Ihnen ankommen ( wir müssen noch das alternative Lot für den Angiopoietin 1 aus Japan einfliegen lassen).

Thank you for contacting us.

I have attached the results of the sequence alignment between mouse and human angiopoietin 1.

The link to our IHC-P protocol is as follows:
https://www.abcam.com/index.html?pageconfig=resource&rid=11384

The file is also attached for your convenience.

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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Thank you for taking the time to complete our questionnaire and get back to me.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

Reviewing this case, I would like to offer some suggestions to help optimise the results from ab8451. I would also appreciate if you can confirm some further details:

1. Please provide the order number and date of purchase.

2. In order to help me assess the results:

What conditions are being using when there is no signal? What was changed afterwards to improve try and improve the results?

What conditions are being used when there is nuclear staining? What was changed afterwards to improve try and improve the results?

What conditions are being used when there is high background? What was changed afterwards to improve try and improve the results?

3. It would be beneficial if you are able to provdie some images? I am sorry you have had difficulty submitting these. It may be that the file size is too large, you could try compressing and sending them as a PDF to see if this works. If you are still having difficulty sending these, please do not hesitate to try sending them to my personal email address kate.hayes@abcam.com.

Please send any text reply by direct reply to this email address (technical@abcam.com) and images to my personal address.

4. I can suggest to try different times for antigen retreival as this can sometimes require some optimization if this has not already been tried.

5. Is the anti rabbit kit and detection kit working well with other primary antibodies?

6. The samples may be varying in quality, especially if they have been collected over so many years at the hospital. Is there any opportunity to try newer samples prepared over the last few weeks?

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

Thank you for taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody and we are happy to help.

I would like to reassure you that this antibody is tested and covered by our 6 month guarantee for IHC-P and human samples. In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily. Once we have this information, I hope to be able to provide some suggestions as requested.

I would appreciate if you could also provide an image which would help us to assess the results.

Thank you for your time and cooperation. We look forward to receiving the completed questionaire.
Order Details
Antibody code:
Lot number:
Purchase order number
or preferably Abcam order number:
General Information
Antibody storage conditions (temperature/reconstitution etc)
Description of the problem (high background, low signal, non-specific satining etc.)
Sample (Species/Tissue/Cell Type/Cell Line etc.)
Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)
Antigen retrieval (Enzymatic method, Heat mediated technique etc.)
Permeabilization step
Blocking conditions (Buffer/time period, Blocking agent etc.)
Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Detection method
Positive and negative controls used (please specify)
Optimization attempts (problem solving)
How many times have you tried the IHC?
Have you run a "No Primary" control?
Yes No
Do you obtain the same results every time?
Yes No
What steps have you altered?
Additional Notes
We would appreciate if you are also able to provide and image which woudl help us to assess the results

Thank you for getting back to us. Unfortunately, xxx is away from the office today so I will deal with this enquiry.

I think what xxxx was suggesting was to perform a "no primary" control, in order to determine if the secondary antibody was making any contribution to the non-specificity observed, as well as a sample known no to express Angiopoietin 1. Unfortunately, we do not have this to provide.I would suggest your customer perform this as well as the additional suggestions Kate made:

1. I can suggest to try incubate overnight 4oC which can often provide more efficient and specific staining. I can also recommend to try a lower dilution, 1:2000 to reduce the non specific staining

2. Could you confirm the wash steps? I can suggest to wash 4 times for 5 minutes in PBS containing 0.2% Tween at the appropriate wash steps if this has not already been tried. Also, changing from PBS to TBS can provide a more stringent wash.

Additionally, could you confirm the following:

1. Could you confirm if the current vial of secondary antibody is working well with other primary antibodies?

2. Could you confirm if a loading control staining has been included to help asses the quality of the samples?

Many thanks for your continued assistance.

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

I would like to reassure you that ab8451 is tested and covered by our 6 month guarantee for use inWB and human and mouse samples. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.

Regarding the results, there should be a single band at 57 kDa (sometimes there is a higher band at around 75 kDa which indicates glycosylated protein, but your results do not show this). Reviewing this case, I would like to offer some suggestions to help optimize the results. I would also appreciate if you can confirm some further details to assist in my investigation of this case.

1. I can suggest it would be beneficial to consider including an endogenous negative control samples. Could you confirm if this has been tried?

2. Could you confirm if the current vial of secondary antibody is working well with other primary antibodies?

3. I can suggest to try incubate overnight 4oC which can often provide more efficient and specific staining. I can also recommend to try a lower dilution, 1:2000 to reducethe non specific staining

4. Could you confirm the wash steps? I can suggest to wash 4 times for 5 minutes in PBS containing 0.2% Tween at the appropriate wash steps if this has not already been tried. Also, changing from PBS to TBS can provide a more stringent wash.

5. Could you confirm if a loading control staininghas been includedto help asses the quality of the samples?

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

Thanks again for the additional data. I think there are a few confounding factors here that make the western blot data difficult to interpret confidentally: 1. I agree with you that the intense 40kDa band is likelyb-actin in both blots, but we can not be certain. 2. There also may be bleed-through from the first anti-angiopoietin blot to the second angiopoietin blot. 3. Pig angtiopoietin-1 has technically not been tested previously with either ab8451 or ab95230, so the specificity of these antibodies with respect to pig samples is not known. 4. The expression pattern ofangtiopoietin-1 in pig kidney tissue, including any alternate forms or glycosylated forms, are not known, at least based on my brief literature search. You may consider treating your samples with or without PNGase-F, an endoglycosylase which would remove complex oligosaccharides from N-linked glycoproteins. If treated samples show a band with a smaller molecular weight, this suggests the detected protein was gylcosylated and likely angtiopoietin-1. I look forward to hearing if these suggestions prove useful.

Thanks for the additional information. A couple more questions... 1. Are one or more of the samples from the blot considered a negative control, in that it is known to not express Angiopoietin 1? 2. After stripping but before reprobing, was it determined that there was no residual ECL signal remaining from the previous blot? Thanks again!