Anti-beta 1 Adrenergic Receptor antibody (ab3442)

Thank you for bringing this to our attention. The two bands you are seeing might represent unphosphorylated (lower band) and fully phosphorylated (upper band) receptor but that is unlikely if the difference in sizes is 5 kDa and you are seeing just two bands. There are at least three known phosphorylation sites. There is also the possibility of glycosylation which would cause a portion of the protein to run more slowly than the unmodified portion.

We have not tested this antibody against mouse brain samples, only the cell lysates listed in the caption of the western blot image on the datasheet. The result shows primarily a single band at the 50 kDa marker, and a very light band at 55 for two or three cell lysates. Other antibodies raised against the receptor, for instance ab85037, tested against mouse spinal cord, show just a single band at about 55 kDa. I suspect that one of the bands you see is unrelated to the receptor. We can send a replacement, for instance ab85307 but first I would like to see if there is something I can suggest to improve your ab3442 result.

Can you please send a file of your blot image, and a few details of your protocol? In particular, how was the sample prepared? Did the prep include protease inhibitors?

We have another report of a band appearing at 57 kDa only when the blot is blocked with BSA. When blocked with 5% milk, the band is not present. How did you block the membrane?

Finally, what was the concentration or dilution of the antibody? Have you stained blots of these samples with any other antibodies, and were the results as expected? I look forward to your reply.

Thank you very much for your inquiry. I apologize for the long delay of my answer.

Firstly, thank you very much for having provided such a nice summary, including the data and the protocol steps. This makes it easier for us, to understand the problem.

I have looked at the data for ab3442 and would like to raise the following points:

1.) When looking at the data you provided, the first thing which comes to my mind is, whether these are not degradation products. Indeed, multitransmembrane protein are very sensitive to heat and can break. Have you heated the samples before you have loaded them on the gel? If yes, I can recommend to test also unheated samples and samples heated just for 5 or 10 minutes at 70°C.

2.) Have the samples been reduced as well?

3.) In order to avoid degradation by proteases as well, have you held the samples always on ice and added protease inhibitors to the RIPA buffer?

4.) I actually can confirm that thelysis buffer you are using (RIPA) should be suitable for membrane proteins.

5.) To my knowledge there are not isoforms described yet for this protein.

6.) The band at 57kD absolutely could be the right band. Depending on the buffer system and the molecular weight marker, there are always slight variation of the apparent molecular weight. Additionally, the Beta-1 adrenergic receptor can also be glycosilated, which increases its apparent molecular weight. http://www.uniprot.org/uniprot/P08588

If with these recommendations we do not resolve the problem, we will replace this antibody for you according to our guarantee. I am however optimistic, that not heating the samples could improve significantly the results. Please let me know how you are getting on and if you have any questions or concerns. I look forward to hear back from you.

Thank you for taking time to complete our questionnaire and for contacting us. I am sorry to hear 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. I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful.

Having reviewed the protocol details, I believe this product should have given satisfactory results. It appears that you may have received a faulty vial.

I apologize for the inconvenience and am pleased to offer you a free of charge replacement, credit note, or refund in compensation. Unfortunately, we only have the same lot of ab3442 in stock at present to offer. Alternatively I can offer the antibody ab85037. This has shown to work well with mouse spinal chord tissue as well as ICC staining of SHSY5Y cells as displayed on the datasheet of this product.

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

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 wouldappreciate it if you could confirm some further details:

1. Has the secondary antibody and Ecl reagent been used successfully in other blots?

2. Has a loading control been performed with the samples?

3. How long was the membrane exposed for?

I would like to offer some suggestions to help optimize the results from ab3442.

1. When testing our antibodies, our lab uses 5% BSA as a blocking reagent, so I recommend switching to this instead of milk. For unknown reasons some antibodies give stronger, more specific signals on blots blocked with BSA instead of milk, so doing this may improve the results you are seeing, and reduce the non-specific bands. An example of the above is the western blot obtained with the Abcam GAPDH antibody ab9385 : https://www.abcam.com/gapdh-antibody-hrp-loading-control-ab9385.html#GAPDH-Primary-antibodies-ab9385-3.jpg (or use the following: https://www.abcam.com/ab9385).BSA should then also be used in the dilutionbuffer of the antibodies instead of milk.

2. I would suggest reducing the amount of protein loaded onto the gel. 20 ug of protein should be sufficient. I would also suggest heating the sample to 70°C for 10 minutes instead of boiling the sample as the target is a membrane bound protein which tend to form aggregated if boiled.

3. I would also strongly suggest performing a "no primary" control in order to deduce if this non-specificity is due to the secondary antibody or the blocking agent.

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.

Thank you very much for your interest in our adrenergic Receptor antibodies. These are the alternatives we could recommend: Anti-beta 1 Adrenergic Receptor antibody (replacement for ab3546): The rabbit polyclonal ab3442 which is guaranteed to work with a number of species including mouse and rat in Western blot and Immunocytochemistry/Immunofluorescence (ICC/IF). To our knowledge, ab3442 has not been tested in Immunhistochemistry on frozen sections (IHC-Fr). Therefore, I can offer a discount off a future purchase if you buy it now, test it in IHC-Fr and submit feedback to us in the form of an Abreview. It doesn’t matter whether the Abreview is positive or negative, we would just really like to receive your feedback. The discount would be to the value of 1 free primary antibody. Please contact us again for more detailed information if you are interested. Click here (or use the following: https://www.abcam.com/index.html?datasheet=3442). Anti-alpha 1d Adrenergic Receptor antibody (replacement for ab12923): The rabbit polyclonal ab84402 which is guaranteed to work with a number of species including human in Western blot and ELISA. This antibody has not been tested in Immunhistochemistry on paraffin-embedded sections (IHC-P). Again, if you would like to test this, you are eligible for our testing discount program if the antibody has not yet been purchased. Click here (or use the following: https://www.abcam.com/index.html?datasheet=84402). Anti-alpha 1c Adrenergic Receptor antibody (replacement for ab12935): The rabbit polyclonal ab96783 or the mouse monoclonal ab87990 which are guaranteed to work with a number of species including human in Western blot and ELISA. These antibodies have not been tested in Immunhistochemistry on paraffin-embedded sections (IHC-P) and here you are eligible for our testing discount program. Click here (or use the following: https://www.abcam.com/index.html?datasheet=96783). Click here (or use the following: https://www.abcam.com/index.html?datasheet=87990). The terms and conditions applicable to these offers can be found here: www.abcam.com/collaborationdiscount. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.