Anti-ATF6 antibody [70B1413] - ChIP Grade (ab11909)

one more question about ab31369-- what does it specifically recognize near the C terminus? could it recognize STAT1 that is cleaved at aspartic acid 694? (lower band in earlier attachment seems unlikely to be STAT1 beta since it may not respond to IFNg at all)
Thank you for the update. I had a few more questions and would appreciate any information you can provide.

ab31369:Does this detect total STAT1 (alpha and beta forms, and more importantly, with or without phosphorylation?)

ab30645: Does this only detect STAT1pY701 and definitely not STAT1pS727? What about STAT1 phosphorylated at both sites?

I've attached some blots, including the image below (STAT1 +/- phosphorylation bands range from ˜85 to 100kDa).Is it possible that the:

top bands are STAT1 alpha phosphorylated at Y701

middle (faint) bands are some sort of an isoform

lower bands are either 1) STAT1 beta (or alpha (less likely due to MW?)) phosphorylated at only S727 or 2) STAT1 alpha phosphorylated at both Y701 and S727?

ab11909: Under certain protein isolation conditions, is it possible to lose inactive ATF6 and only detect cleaved ATF6? What would those conditions be and how would the full-length ATF6 be lost?

Please let me know if you have any questions. Thanks much for your help in advance.

ANSWER:

Thank you for your emails and for your patience while I've looked into these questions.

I've heard back from the lab regarding the specificity of ab31369. The antibody should detect both the alpha and beta forms, thoughwe haven't specifically tested whether it reacts with the phosphorylated protein too. There might be some reactivity with the phosphoprotein, but we would expect it to be weaker than reactivity with the non-phosphorylated protein. Based on our conversation last week and the images you sent, it looks like the antibody is reacting quite strongly with the phosphoprotein. The lab might be running some tests to determine this, and I'll keep you posted on anything else that we find out. I'll also need to check whether the antibody could detect STAT1 cleaved at residue 694, as I don't have the full immunogen seequence in my notes.

Ab30645 is specific for STAT1 phosphorylated at Y701, and I would expect it to react with protein phosphorylated at both Y701 and S727. Some phospho-specific antibodies show weak cross-reactivity for other phospho-proteins, so it is possible that ab30645 would weakly cross-react with protein only phosphorylated at S727. I'm not sure that this has been specifically tested by the lab, but I'll check with them and get back to shortly with that information.

Regarding the images, based on molecular weights I would expect the higher band to be the alpha isoform phosphorylated at both Y701 and S727, while the lower bands would be either the beta isoform (phosphorylated or not) or alpha isoform only phosphorylated at Y701. Phosphorylations eachtend to add some weight to the protein. I think further studies might need to be done in order to know this for sure, but that seems to be the most likely explanation. There are too many possible post-translational statesat similar molecular weights in order to know for sure what each band represents based on band size.

For ab11909, I may not be understanding the question as intended, but the full-length inactive ATF6 protein is cleaved by proteases during ER stress, so I would expect this form of the protein if the cells are under this type of stress. The full-length ATF6 most likely isn't completely lost, butthe cleaved form could certainly be more abundant in the sample and the inactive ATF6 at undetectable levels. It is also possible for proteases in the samples to cleave the protein during protein isolation/sample preparation but this would generally result in several lower molecular weight bands on the Western blot. If the samples are not treated with enough protease inhibitors (or the right mix of protease inhibitors) or were not kept on ice during preparation, it is possible for this to occur. If the samples were treated correctly, and the cells were under ER stress, than I expectthat specific proteases just cleaved the protein to yield the active form. Please let me know if I misunderstood the question or if there is more information that you're seeking.

I hope this information will be useful, but please let me know if you have further questions or if there is anything else that we can do for you. I'll be back in touch once I have more information from the lab about the STAT1 antibodies.

I'm in the process of blotting with the recommended condition right now and have my fingers crossed.
I've been having trouble getting another antibody optimized as well and wonder if you might be able to provide some additional information about the conditions used for it as well. It is ATF6 product #ab11909.
Thanks again for all of your help!

ANSWER:

Thank you for your reply.

For ab11909, the antibody was used at 3 ug/mL (though we recommend 1-5 ug/mL depending on the expression in your sample). Protein was blotted onto a PVDF membrane which was then stained with Amido black to confirm transfer. The membrane was blocked with 5% nonfat dry milk in TBST for 30-60 minutes. The primary antibody was incubated in 1% milk/TBST overnight at 4C. The membrane was washed 5x with TBST then treated with secondary antibody (1:1000-1:10000) for an hour at 4C in 1% milk in TBST. After 5x washes with TBST, the protein was detected with pico ECL substrate.

Please let me know if this information is helpful or if there is anything else that you need, and I'll be happy to help. Have a wonderful day.

Not working in ICC or WB with mouse motor neurons.

ANSWER:

Thank you for you call today and letting us know about the problem with ab11909 in ICC and WB.

As we discussed, I am sending a free of charge vial of ab37149 on the order ***, which should arrive later today or tomorrow. Please keep me updated about how this replacement antibody works.

I look forward to hearing from you. Let me know if you have any questions or if there is anything else that we can do for you.

Vielen Dank für Ihre ausführlich Mail und die vielen Hinweise. Hinsichtlich des ORP150 wären wir sehr an dem Blocking Peptid interessiert. Wäre es in diesem Fall möglich, dass Sie uns dieses kostenfrei zukommen ließen, damit wir diesen sehr wichtigen Antikörper in unserem Labor für humane Proben etablieren könnten? Ihre Anmerkungen bezüglich des ATF6 gesamt - und des aminoterminalen Antikörper habe ich mit großem Interesse gelesen und werde mit diesen weiter an der Etablierung des Antikörper arbeiten, allerdings stellt sich mir die Frage, warum im Datenblatt von Abcam für den ab65838 folgendes steht: "Predicted molecular weight: 75 kDa" Ich gehe davon aus, dass ungeschnittenes ATF6 ein Molekulargewicht von 90 kDa aufweist, siehe auch ab11909, und das beim Schneiden ein Fragment mit 50 kDa und eines mit 40 kDa entsteht - wo tauchen dann aber die 75 kDa auf?

ANSWER:

Vielen Dank für Ihre Antwort und für diese weiteren Informationen. 

Bitte entschuldigen Sie die Verzögerung. Ich habe nun eine kostenfreie Lieferung des Blockpeptides für ORP150 arrangiert. Sie hat die Referenznummer xxxxx und sollte in den nächsten Tagen bei Ihnen ankommen. 

Die Angabe des erwarteten Molekulargewichts bezieht sich ausschließlich auf den Proteinanteil von ATF6. Unter der SwissProt-Identifikation P18850 (http://www.uniprot.org/uniprot/P18850 ) wird diese mit 75 kDa angegeben; die Schnittstelle befindet sich bei Aminosäure 419/420, also nach etwa 2/3, so dass ein ca 50 kDa und ein ca 25 kDa Fragment entstehen. Allerdings ist zu beachten, dass ATF6 recht stark glykosyliert sein kann, was die Laufeigenschaften des Proteins in einem Gel beeinflussen kann. Dadurch ließe sich auch erklären, dass ab11909 eine Bande von über 90 kDa für das ungeschnittene Fragment detektiert. Das 36 kDa-Fragment wurde leider nicht charakterisiert; es kann sich jedoch um ein ATF6-Abbaufragment handeln (z.B. das kleinere 25 kDa-Fragment plus Glykosylierung).

Für diese nicht ganz eindeutigen Angaben möchte ich mich bei Ihnen entschuldigen. Bei den Angaben auf unseren Datenblättern sind wir leider auf die Angaben und Abbildungen angewiesen, die uns das herstellende Labor zur Verfügung stellt. Daher haben wir nicht immer alle nötigen Informationen zu den detektierten Banden bzw. den Bedingungen, unter denen die Zellen kultiviert/behandelt wurden.

Ich hoffe dennoch, dass diese Informationen Ihnen weiterhelfen. Bitte zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.

Thanks so much for all your help in this. The "order reference number" is ###### and invoice number is ###########. Please let me know if you need any further information. Thanks so much and have fantastic day!  

ANSWER:

Thank you for your email.

Your credit note ID is ######. I am sorry that this antibody did not perform as stated on the datasheet. I have asked our accounting department to issue a credit note for you, which can be redeemed against the invoice of a future order by passing it on to your purchasing department. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used. If you have questions on how to use the credit note, please contact our accounting department. Our accounting department can be contacted by email at us.credits@abcam.com or by telephone using the information at the Contact Us link in the top right corner of our website. Please refer to the credit note ID in any correspondence with our accounting department. The credit note ID is for your reference only and does not automatically guarantee the credit. I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service, should you require further expert advice.