Anti-APAF1 antibody (ab2001)

Thank you for your suggestion and co-operation. I would appreciate if you kindly replace the antibody so that I can carry out my experiment. I look forward for your reply,
Best,

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:

Order number: 1083546

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.

I have tried your following suggestions, correct secondary antibody is used. the vial is correctly working with other primary antibody. I can send you the DAB stained picture,let me know whether it will help you.
best,

ANSWER:

Thank you for your message and for kindly providing this further information.

I am sorry to hear the suggestions made have not improved the results on this occasion. I appreciate the time you have spent on these experiments and would be pleased to arrange a free of charge replacement or credit note in compensation.

I look forward to hearing from you with details of how you would like to proceed.

Product code: 2001
Lot number: GR 26336-2
Inquiry: Hello, I had contacted the technical help desk earlier for my problem with immunoblot. I tried the suggestions and I got bands, in one blot i gt band around 130 kDa, with some non specific bands but in subsequent blot i got single band around (range between 55-75 kDa). I checked with the literature and i got variation in band size of Apaf1. Please provide me information of the variation of Apaf 1 size. I am looking for Apaf1 in apoptotic brain tissues. your help would be highly appreciated. best,

ANSWER:

Thank you for providing this further information.

I appreciate your cooperation and understand your concerns. It is regrettable the results have not been succesful. I have traced your previous corresopndence and reveiwed this case.

With regards to the band sizes expected for PAF1 protein in WB, the Uniprot protein database lists 6 isoforms of different sizes:

http://www.uniprot.org/uniprot/O14727
Isform 1 142 kDa
Isoform 2 141 Kda
Isoform 3 136 Kda
Isoform 4 137 kDa
Isoform 5 133 kDa
Isoform 6 37 kDa

Unfortunately, none of these would match the bands at 55 - 75 kDa that you are seeing. I can confirm this antibody has not specifically been tested on brain samples in WB and regrettably, I am having difficulty confirming which isoforms are known to be expressed in the brain with a literature search. I could just find the following article: http://www.pnas.org/content/104/52/20820.full that mentions normal brain has little Apaf-1 and tumors have a higher level, but unfortunately nothing specific to which isoforms are in the brain.

Could you confirm if any samples from other tissue have been included as a positive control? I can suggest this would be highly beneficial in this case.

In order to help with our investigation of this case before deciding how to proceed, I would appreciate if you are able to provide more detail as requested in the previous email. This information will also be helpful to our quality monitoring.

1. Please confirm the Abcam order reference number and date of purchase

2. For the secondary antibody, I can suggest an anti-rabbit secondary would be required to detect the rabbit polyclonal primary. An anti-goat would not detect rabbit polyclonal. If the correct secondary is being used, I can sugest it is also worth considering if the current vial is working well with other primary antibodies?.

3. Try a higher concentration of antibody to increase the signal. Try 1:1000. Incubate overnight 4oC.

4. Could you confirm which lysis buffer was used? I can recommend to lyse the cells in RIPA lysis buffer if this has not already been tried, before adding SDS loading buffer such as Laemmli to reduce and denature the samples. This should provide a more suitable protein preparation.

I would appreciate if you could also provide an image, including molecular weight markers, which would help us to assess the results.

I will be pleased to provide a free of charge replacmenet or credit note if the antibody is not working with the suggestions provided.

Thank you for your time and cooperation. We look forward to receiving the requested information and results from the positive control.

LOT NUMBER GR 26336-2
ORDER NUMBER -- NOT SPECIFIED --
DESCRIPTION OF THE PROBLEM No signal
SAMPLE CYTOSOLIC FRACTION OF MALE WISTER RAT BRAIN HOMOGENATE- , PC12-LYSIS PROTEIN, NEURA 2A LYSED PROTEIN
PRIMARY ANTIBODY 1:2000
DILUTION IN 0.03%BSA IN TBST
DETECTION METHOD ecl
POSITIVE AND NEGATIVE CONTROLS USED no positive control
ANTIBODY STORAGE CONDITIONS -80c
SAMPLE PREPARATION DID HEATING AT 94 DEGREES FOR 4 MINUTES WITH DYE
AMOUNT OF PROTEIN LOADED 100 microgram
ELECTROPHORESIS/GEL CONDITIONS 8% gel
TRANSFER AND BLOCKING CONDITIONS BSA BUFFER, OVERNIGHT
SECONDARY ANTIBODY ANTI GOAT ANTIBODY 1:10000 DILUTION
HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 5
HAVE YOU RUN A "NO PRIMARY" CONTROL? No
WHAT STEPS HAVE YOU ALTERED? incubation PERIOD of primary antibody increased, 4 C, RT tried

ANSWER:

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 ab2001 is tested and covered by our 6 month guarantee for use in WB and rat 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.

Reviewing this case, I would like to offer some suggestions to help optimise the results from ab2001. I would also appreciate if you can confirm some further details:

1. Please confirm the Abcam order reference number and date of purchase

2. For the secondary antibody, I can suggest an anti-rabbit secondary would be required to detect the rabbit polyclonal primary. An anti-goat would not detect rabbit polyclonal. If the correct secondary is being used, I can sugest it is also worth considering if the current vial is working well with other primary antibodies?.

3. Try a higher concentration of antibody to increase the signal. Try 1:1000. Incubate overnight 4oC.

4. Could you confirm which lysis buffer was used? I can recommend to lyse the cells in RIPA lysis buffer if this has not already been tried, before adding SDS loading buffer such as Laemmli to reduce and denature the samples. This should provide a more suitable protein preparation.

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.

This email is additional information for my previous email whichi subject is "Question about antibody and peptid".

I forgot to ask you how pure they are and which method these peptides have been purified by.

Thank you for your cooperation.

ab2001, HREALEKDIKTSYIMDHC ab7346, SSPIYDINYYTSEPC ab3430, MSDFDSNPFADPDLNNPC ab3346, MSAPAQPPAEGTEGAAPGC ab3347, MSATAATVPPAAPAGEGGC

Thank you for your cooperation

ANSWER:

Thank you again for your enquiry. Regarding ab4969, ab4957, and ab4958 (the peptides corresponding to the antibodies you indicated), they are all greater than 95% pure, and they were purified via HPLC.

If you have any further questions, please contact us again.