Anti-Ryanodine Receptor antibody [34C] (ab2868)

Subject: Re: Ryanodine Receptors Abupdate

Please check for mea midsagittal section of rat or mouse cerebellum. .Does this antibody stain small neurons in the granular layer of the caudal cerebellar vermis?
Regards,

ANSWER:

Thank you for email.

This antibody is very well charaterized. We know this antibody detects RyR-3 in mouse cells and by Western blot, this antibody detects a 565 kDa protein representing RyR from rat skeletal muscle extracts.

The RyR-3 detected ion mouse is mainly expressed smooth muscle and the brain (striatum, thalamus and hippocampus).

We do not have any data about rat or mouse cerebellum and also donot know if this antibody will stain neurons in the granular layer of the caudal cerebellar vermis.

If there is evidence that RyR-3 is expressed inneurons in the granular layer of the caudal cerebellar vermis in mouse than I can recommend this antibody. But we do not want to suggest a product that might be unsuitable for your purposes and therefore recommend a literature search.

I am sorry I could not answer your questions on this occasion and hope this information is nevertheless helpful.

Thank you very much for your kind, prompt reply. Do you have other specific phospho antibodies as well?

ANSWER:

Thank you for your reply.


We have a number of phospho specific antibodies in our catalog, but ab59225 is the only phospho specific antibody we have to Ryanodine Receptors.


In order to determine specifically which residues are phosphorylated, you can perform an IP with an antibody to total ryanodine receptor (such as ab2868) and analyze the product in a mass spectrometer.


Another possibility would be to perform a sandwich ELISA using an antibody to total ryanodine receptor for capture and an antibody to a phosphorylated residue for detection. While this approach would not tell you which residues specifically are phosphorylated, you would be able to differentiate between phosphoserine (ab6639), phosphothreonine (ab9337), and phosphotyrosine (ab9319).


I hope this helps, please let me know if you need any additional information.

Haben Sie Tips zum Blotten diese 565 kDa Proteins? Haben schon einiges probiert, funktioniert nicht. Die meisten Marker gehen nur bis 460 kDa.

ANSWER:

Vielen Dank für Ihren Anruf. Das Labor hat mir mitgeteilt, dass für den Western blot ein 6- oder 8%iges Acrylamidgel empfohlen wird und der Transfer 'wet' (d.h. 'not fast or semi-dry') erfolgen sollte. Dies kann bei 90 Volt für 2 Stunden oder niedrigerer Spannung über Nacht geschehen, wie Sie schon vorgeschlagen hatten. Wichtig sind auch SDS im Transferpuffer in einer Endkonzentration von 0.1%, weil dies die Präzipitation großer Proteine verhindert. Eine geringe Methanolkonzentration im Transferpuffer verbessert ebenfalls den Transfer. Außerdem handelt es sich beim Ryanodinrezeptor um ein Protein mit mehreren Transmembrandomänen. Daher könnten bessere Ergebnisse erzielt werden, wenn die Proben nicht bei 95-100ºC, sondern nur bei 60-70ºC aufgekocht werden, und statt Beta-Mercaptoethanol im Ladepuffer besser DTT verwendet wird (http://www.abcam.com/index.html?pageconfig=resource&rid=11379#A6: 6. Preparation of samples for loading into gels; siehe auch Anhang). Der Antikörperklon [34C] wurde schon oft in der Literatur zitiert, unter anderem in Walton et al (PMID 1645737, siehe Anhang), und ich bin mir sicher, dass Sie darin noch detaillierte Protokollbeschreibungen finden. Die Transferbedingungen in dieser Publikation waren anfänglich 100 V für 2 Stunden, danach 40 V über Nacht in 10 mM 2[N-cyclohexylamino] ethan-suifonic acid, pH 9.6 und 10% Ethanol. Auch die folgenden Publikationen könnten hilfreiche Informationen enthalten: U. Schmidt, et. al. Restoration of Diastolic Function in Senescent Rat Hearts Through Adenoviral Gene Transfer of Sarcoplasmic Reticulum Ca2+-ATPase. Circulation, Feb 2000; 101: 790 - 796. http://circ.ahajournals.org/cgi/reprint/101/7/790 Z. Liu, et al Localization of a Disease-associated Mutation Site in the Three-dimensional Structure of the Cardiac Muscle Ryanodine Receptor. JBC Dec 2005 Manuscript M505714200. http://www.jbc.org/cgi/reprint/M505714200v1 G. Anyatonwu, et al. Organic Cation Permeation through the Channel Formed by Polycystin-2. JBC Jun 2005 Manuscript M504359200. http://www.jbc.org/cgi/reprint/M504359200v1 Marisa Brini, et al. Ca2+ Signaling in HEK-293 and Skeletal Muscle Cells Expressing Recombinant Rynaodine Receptors Harboring Malignant Hyperthermia and Central Core Disease Mutations. JBC Feb 2005 Manuscript M410421200. http://jbc.org/cgi/reprint/M410421200v1 D. Bare, et al. Cardiac Type 2 Inositol 1,4,5-Triphosphate Receptor: Interaction and Modulation by Calcium/Calmodulin-Dependent Protein Kinase II. JBC Feb 2005 Manuscript M414212200. http://www.jbc.org/cgi/reprint/M414212200v1 P. Koulen, et. al. Differentially Distributed IP3 Receptors and Ca2 Signaling in Rod Bipolar Cells. IOVS 46(1):292-298, 2005. http://www.iovs.org/cgi/reprint/46/1/292 B. Xiao, et. al. Isoform-dependent Formation of Heteromeric Ca2+ Release Channels (Ryanodine Receptors). JBC 277(44):41778-41785, 2002. http://www.jbc.org/cgi/reprint/277/44/41778 P. Aracena-Parks, et. al. Identification of Cysteines Involved in S-Nitrosylation, S-Glutathionylation, and Oxidation to Disulfides in Ryanodine Receptor Type 1. J. Biol. Chem., Dec 2006; 281: 40354 - 40368. http://www.jbc.org/cgi/reprint/281/52/40354 Wie am Telefon erwähnt, möchte ich Sie gerne auf unsere Optiblot-Gelelektrophorese-Sparte aufmerksam machen (http://www.abcam.com/Optiblot). Die Gele, die wir neu in unseren Katalog aufgenommen haben, sind aufgrund einer neuen Polymerisationsstruktur besonders stabil, was gerade bei einer niedrigen Konzentration von 4-8% von Vorteil sein kann (ab119204 bzw. ab119208, siehe unten). Leider haben wir derzeit keinen High Molecular Weight-Marker bis 565 kDa erhältlich; allerdings gibt es im Moment zu jeder Optiblot-Bestellung einen Marker kostenlos (ab123069). Click here (or use the following: http://www.abcam.com/index.html?datasheet=119204). Click here (or use the following: http://www.abcam.com/index.html?datasheet=119208). Ich hoffe, diese Informationen sind hilfreich für Sie. Wir würden jedoch gerne mit Ihnen in Kontakt bleiben, um sicherzustellen, dass Sie zufriedenstellende Ergebnisse mit dem Antikörper erzielen, wie es unsere Abpromise garantiert (http://www.abcam.com/Abpromise). Bitte lassen Sie mich daher wissen, ob ich Ihnen helfen konnte und zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.

Dear Sir/Madam, I have a question about your antibody ab2868 (Anti-Ryanodine Receptor, RyR). In the description of the antibody specificity it says 'Immunohistochemical staining of RyR in chicken brain results in intense staining of cerebral Purkinje neurons.' 1. I suppose you mean cerebellar, rather than cerebral Purkinje neurons. 2. Is this only true for chicken or do you have data for other species also (mouse, rat, human)? Thank you very much in advance. Yours faithully    

ANSWER:

Thank you for your inquiry.

1. Thank you for making is aware of this typo. This has been corrected.

2. We only are aware of a publication for chicken where this clone of antibody was used:

J Cell Biol. 1991 Jun;113(5):1145-57. "Ryanodine and inositol trisphosphate receptors coexist in avian cerebellar Purkinje neurons." Author(s): Walton PD, Airey JA, Sutko JL, Beck CF, Mignery GA, Südhof TC, Deerinck TJ, Ellisman MH

Unfortunately, we do not have any in-house data on other species cerebellar Purkinje neurons. This clone is widely used and we might not be aware of all publications with.

I hope this information is helpful and wish you good luck with your research.

LOT NUMBER GR30154-1

ORDER NUMBER PO-10699

DESCRIPTION OF THE PROBLEM

No bands

SAMPLE

RyR2 stably expressed in HEK293 cells. Used many time before. Expression confirmed using functional assays using same cells.

PRIMARY ANTIBODY

34C, Abcam, ab2868, 1:1000, PBS with 20% FBS, 2-4hr, 3x 5min PBS-Tween

DETECTION METHOD

Pico ECL (Pierce)

POSITIVE AND NEGATIVE CONTROLS USED

Negative controls: cells not expressing RyR2

Positive control: None, functional assay shows cells must express RyR2

ANTIBODY STORAGE CONDITIONS

4oC

SAMPLE PREPARATION

Cells lysed using a CHAPS based buffer, protease inhibitors added, tried both boiling for 5mins and 30min at 55oC. Also tried immunoprecipitation with the ab and no bands could be detected by total protein stain.

All methods used previously http://www.biochemj.org/bj/404/0431/bj4040431.htm

AMOUNT OF PROTEIN LOADED

2 to ~200ug per lane

ELECTROPHORESIS/GEL CONDITIONS

Reducing, 6%

TRANSFER AND BLOCKING CONDITIONS

Wet transfer 18hrs, constant V (45V) at 4oC, tried nitrocellulose and PVDF, blocked with 5% milk in PBS-tween

SECONDARY ANTIBODY

Goat anti-mouse-HRP (ab97023), goat, PBS, 1:20000, 90min, 3x 5min PBS-Tween

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 10

HAVE YOU RUN A "NO PRIMARY" CONTROL? No

DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED?

Tried loading different amounts of protein, altered incubation length, tried immunoprecipitating protein to increase loading

ADDITIONAL NOTES

I have successfully used this Ab clone with the cell line many times before with no problems using the conditions described, eg. http://www.biochemj.org/bj/404/0431/bj4040431.htm

I also get no signal when using it for ICC (see other other technical form fill in online)

ANSWER:

Thank you for your enquiry regarding ab2868 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.

This antibody is cited in many publications and Abreview so in order to understand the problem better I have few more questions to ask. I would appreciate if you can kindly answer as this will help us in quality control of the lot you received.

- Could you let us know the species of fragment expressed in HEK293 cells?

- Regarding the information provided in additional note; have you used same antibody from Abcam before or from different source. Were the lysates and cells similar to the previous experiments?

- In WB experiments with transfected lysates, positive controls are always good. Have you used any? We can recommend using lysates of PC12 cell line or rat skeletal muscle tissue lysates.

- Could you specify how long the antibody was stored at 4C.?

Looking forward to hearing from you soon.

PS: I have closed CCE3174303 related to ICC; as I will be dealing with both cases in this CCE.