Anti-EDG1 antibody (ab23695)

i confirm the order number for ab23695.
wheter it's possible, i hope you would send me a suitable lysate.
thank in advance.
best regards.

ANSWER:

I have now had confirmation from Prodotti Gianni of the order numbers and have arranged for a human brain tissue lysate to be sent to you. This should show a positive signal for EDG1. It is likely that both the glycosylated and unglycosylated form will be present. You may therefore want to perform the same deglycosylation experiment that you performed with your samples.

In addition to using this tissue lysate, I would also consider implementing the changes I have suggested previously:

1. This is a multi-pass membrane protein and such can be prone to aggregation. This can be reduced by heating to 70 degrees for 10 minutes instead of 95 degrees.

2.Reducing the amount of antibody used from a dilution of 1/100 to 1/250 and 1/500.

3. Trying an alternative blocking agent, I would suggest using 3% BSA as the blocking agent. I would also include 3% BSA in the diluent of the antibodies.

I hope this lysate helps to resolve the problem. If you continue to have problems please do let me know.

Until then, I wish you all the best with your research.

Dear xxxx,
i confirm the order number for ab23695.
wheter it's possible, i hope you would send me a suitable lysate.
thank in advance.
best regards.

ANSWER:

Thank you for getting back to me.

Unfortunately for the emails you have sent to me I could not find the details of your order number in order to organise a positive control lysate for you. If you could provide me with an order number or an approximate date of the order and a delivery address I can arrange this for you. I am sorry if you have already provided this information for me.

I look forward to receiving your reply.

Dear xxxxx,
thank you for your suggestion. Here we have provided the information that you required.
Finally, we would like to know whether it is possible to have an aliquot of cell or tissue lysate to use as positive control for our WB experiments.
Thank you in advance.
Best regards,
xxxxx

ANSWER:

Thank you for providing that additional information. It has greatly helped in our understanding of the problems you have been experiencing.

Having reviewed the protocol details provided I would still advise trying the protocol amendments that I have suggested in my previous email:

The extra band could be caused in part by aggregation of the protein. This is a multi-pass membrane protein and such can be prone to aggregation. This can be reduced by heating to 70 degrees for 10 minutes instead of 95 degrees as you have been doing.

I would also suggest reducing the amount of antibody used from a dilution of 1/100 to 1/250 and 1/500. We often also find trying an alternative blocking agent can have a dramatic effect on the background observed (as is very well illustrated on the datasheet of ab9385). I would therefore suggest using 3% BSA as the blocking agent. I would also include 3% BSA in the diluent of the antibodies.

As well as this I would suggest using slightly less protein, certainly for the patient sample (reduce to 10 ug) and increase that used for the controls HB and HT (to ascertain exactly what bands are picked up in each sample at the same exposure time).

I hope these suggestions help however, 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.

This antibody has been used with human liver microsomes, mouse liver homogenate, pig liver supernatant and rat brain homogenate.We do not have samples available of this, but if you would like I can send youa suitable lysate for you to try. In order toorganise this, could you please confirm your order number for ab23695 (or if you do not have this information, the approximate date of placing the order and the delivery address)?

Many thanks for your cooperation in this matter.

Dear Sir/ Madame,
We have bought from you the anti-EDG1 antibody (ab23695) and we have tested it in WB. Since we have obtained multiple bands at different MW (not affected in number/MW by deglycosilation treatment), we would like to have your opinion about our results and eventual suggestion. Please find attached results and details about specimens and protocol used.
Thank you in advance,
Best regard

ANSWER:

Thank you for your enquiry regarding ab23695 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.

Though you have kindly provided some details and an image, it would be much appreciated if I could get some more information which would help me identify the source of the problem.

In order to be able to provide sufficient support and technical advice, could you provide some further details of the protocol used and complete the following form (attached as a word document).

Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.

Dear Sir/ Madame,
We have bought from you the anti-EDG1 antibody (ab23695) and we have tested it in WB. Since we have obtained multiple bands at different MW (not affected in number/MW by deglycosilation treatment), we would like to have your opinion about our results and eventual suggestion. Please find attached results and details about specimens and protocol used.
Thank you in advance,
Best regard

ANSWER:

Thank you for contacting us and reporting the problems you have encountered in using Anti-EDG1 antibody (ab23695). 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, it indeed appears that the band at ˜60 kDa is not a glycosylated form of the protein. There are however a few suggestions that I can make which may be able to improve on the results. The extra band could be caused in part by aggregation of the protein. This is a multi-pass membrane protein and such can be prone to aggregation. This can be reduced by heating to 70 degrees for 10 minutes instead of 95 degrees as you have been doing.

I would also suggest reducing the amount of antibody used from a dilution of 1/100 to 1/250 and 1/500. We often also find trying an alternative blocking agent can have a dramatic effect on the background observed (as is very well illustrated on the datasheet of http://www.abcam.com/ab9385). I would therefore suggest using 3% BSA as the blocking agent. I would also include 3% BSA in the diluent of the antibodies.

As well as this I would suggest using slightly less protein, certainly for the patient sample (reduce to 10 ug) and increase that used for the controls HB and HT (to ascertain exactly what bands are picked up in each sample at the same exposure time).

If you would be able to confirm your order number and/or lot number I will be able to check if there have been any similar reports previously for the antibody you are using.

I hope these suggestions help however, 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.