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

Mouse monoclonal Ryanodine Receptor antibody [34C]. Validated in WB, IP, IHC, Inhibition, ICC/IF and tested in Mouse, Rat, Sheep, Rabbit, Cow, Dog, Human, Pig, Xenopus laevis, Non human primates.

Overview

  • Product name
    Anti-Ryanodine Receptor antibody [34C]
    See all Ryanodine Receptor primary antibodies
  • Description
    Mouse monoclonal [34C] to Ryanodine Receptor
  • Host species
    Mouse
  • Specificity
    Detects Ryanodine Receptor (RyR)-1 and RyR-2 isoforms. In chickens, this antibody detects the alpha, beta and cardiac isoforms. This antibody also detects RyR-3 in mouse cells. In frog, this antibody detects the alpha and beta isoforms. In fish, this antibody detects the alpha isoform. By Western blot, this antibody detects a 565 kDa protein representing RyR from rat skeletal muscle extracts. In non-mammalian vertebrates, a doublet is seen at 565 kDa representing the alpha and beta isoforms of the receptor. Immunohistochemical staining of RyR in chicken brain results in intense staining of cerebellar Purkinje neurons.
  • Tested applications
    Suitable for: ICC/IF, IHC-P, IHC-Fr, IP, WB, Inhibition Assaymore details
  • Species reactivity
    Reacts with: Mouse, Rat, Sheep, Rabbit, Cow, Dog, Human, Pig, Xenopus laevis, Non human primates
    Predicted to work with: Fish, Amphibian
  • Immunogen

    Other Immunogen Type corresponding to Chicken Ryanodine Receptor. Partially purified chicken pectoral muscle ryanodine receptor.

  • Positive control
    • rat skeletal muscle

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -80°C. Avoid freeze / thaw cycle.
  • Storage buffer
    Preservative: 0.05% Sodium azide
  • Concentration information loading...
  • Purity
    Protein A purified
  • Primary antibody notes
    The Ryanodine Receptor (RyR) is the channel responsible for calcium release from muscle cell Sarcoplasmic Reticulum (SR) and also plays a role in calcium regulation in non-muscle cells. The RyR exists as a homotetramer and is predicted to have a short cytoplasmic C-terminus and 4-10 transmembrane domains. The remainder of the protein, termed the "foot" region, is located in the cytoplasm between the transverse tubule and the SR. Mammalian RyR isoforms are the product of three different genes: RyR-1 is expressed predominantly in skeletal muscle and areas of the brain; RyR-2 is expressed predominantly in heart muscle but also found in the stomach, endothelial cells and diffuse areas of the brain; and RyR-3 is found in smooth muscle and the brain (striatum, thalamus and hippocampus). In non-mammalian vertebrates, the RyR isoforms are termed alpha, beta and cardiac which correlate loosely to the mammalian RyR-1, RyR-3 and RyR-2 isoforms respectively.
  • Clonality
    Monoclonal
  • Clone number
    34C
  • Isotype
    IgG1
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab2868 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ICC/IF Use a concentration of 1 µg/ml.
IHC-P 1/100.
IHC-Fr 1/100 - 1/1000.
IP Use at an assay dependent concentration.
WB 1/5000. Predicted molecular weight: 565 kDa. As RyR is a large protein, we recommend using a 6-8% gel, wet transfering protein overnight at low voltage, adding 0.1% SDS to transfer buffer and reducing methanol to 10% or less.
Inhibition Assay Use at an assay dependent concentration.

Target

  • Function
    Calcium channel that mediates the release of Ca(2+) from the sarcoplasmic reticulum into the cytoplasm and thereby plays a key role in triggering muscle contraction following depolarization of T-tubules. Repeated very high-level exercise increases the open probability of the channel and leads to Ca(2+) leaking into the cytoplasm. Can also mediate the release of Ca(2+) from intracellular stores in neurons, and may thereby promote prolonged Ca(2+) signaling in the brain. Required for normal embryonic development of muscle fibers and skeletal muscle. Required for normal heart morphogenesis, skin development and ossification during embryogenesis.
  • Tissue specificity
    Skeletal muscle and brain (cerebellum and hippocampus).
  • Involvement in disease
    Malignant hyperthermia 1
    Central core disease of muscle
    Multiminicore disease with external ophthalmoplegia
    Myopathy, congenital, with fiber-type disproportion
    Defects in RYR1 may be a cause of Samaritan myopathy, a congenital myopathy with benign course. Patients display severe hypotonia and respiratory distress at birth. Unlike other congenital myopathies, the health status constantly improves and patients are minimally affected at adulthood.
  • Sequence similarities
    Belongs to the ryanodine receptor (TC 1.A.3.1) family. RYR1 subfamily.
    Contains 3 B30.2/SPRY domains.
    Contains 5 MIR domains.
  • Domain
    The calcium release channel activity resides in the C-terminal region while the remaining part of the protein constitutes the 'foot' structure spanning the junctional gap between the sarcoplasmic reticulum (SR) and the T-tubule.
  • Post-translational
    modifications
    Channel activity is modulated by phosphorylation. Phosphorylation at Ser-2843 may increase channel activity. Repeated very high-level exercise increases phosphorylation at Ser-2843.
    Activated by reversible S-nitrosylation. Repeated very high-level exercise increases S-nitrosylation.
  • Cellular localization
    Sarcoplasmic reticulum membrane. Membrane. The number of predicted transmembrane domains varies between orthologs, but both N-terminus and C-terminus seem to be cytoplasmic.
  • Information by UniProt
  • Database links
  • Alternative names
    • Arrhythmogenic right ventricular dysplasia 2 antibody
    • ARVC2 antibody
    • ARVD2 antibody
    • Brain ryanodine receptor calcium release channel antibody
    • Brain type ryanodine receptor antibody
    • Cardiac muscle ryanodine receptor calcium release channel antibody
    • Cardiac muscle type ryanodine receptor antibody
    • CCO antibody
    • Central core disease of muscle antibody
    • HBRR antibody
    • hRYR 2 antibody
    • hRYR2 antibody
    • MHS antibody
    • MHS1 antibody
    • Ryanodine receptor 1 (skeletal) antibody
    • Ryanodine receptor 1 antibody
    • Ryanodine receptor 2 (cardiac) antibody
    • Ryanodine receptor 2 antibody
    • Ryanodine receptor 3 antibody
    • Ryanodine receptor type1 antibody
    • RYDR antibody
    • RYR 1 antibody
    • RYR 2 antibody
    • RYR 3 antibody
    • RYR antibody
    • RYR-1 antibody
    • RyR1 antibody
    • RYR1_HUMAN antibody
    • RYR2 antibody
    • RYR3 antibody
    • Sarcoplasmic reticulum calcium release channel antibody
    • Skeletal muscle calcium release channel antibody
    • Skeletal muscle ryanodine receptor antibody
    • Skeletal muscle type ryanodine receptor antibody
    • Skeletal muscle-type ryanodine receptor antibody
    • SKRR antibody
    • Type 1 like ryanodine receptor antibody
    • Type 1 ryanodine receptor antibody
    • VTSIP antibody
    see all

Images

  • Representative Western blots for Ser(P)2814 and Ryanodine Receptor in control and Cav1.2I/E mutant mouse hearts. Ryanodine Receptor (bottom panel) was detected with ab2868.
  • ICC/IF image of ab2868 stained PC12 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab2868, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • ab2868 staining Ryanodine Receptor (red) in Mouse Skeletal muscle cells at day 10 of agrin-treated differentation by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.25% Triton X-100 in PBS and blocked with 10% serum for 45 minutes at 20°C. Samples were incubated with primary antibody (1/200 in PBS + 3% BSA) for 2 hours at 20°C. An Alexa Fluor® 647-conjugated Donkey anti-goat IgG polyclonal (1/500) was used as the secondary antibody. Nucleus stained blue.

    See Abreview

  • Immunohistochemistry was performed on biopsies of deparaffinized Human skeletal muscle tissue. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:20 with a mouse monoclonal antibody recognizing Ryanodine Receptor ab2868 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

  • ab2868 at a 1/200 dilution staining Ryanodine Receptor in mouse liver tissue sections by Immunohistochemistry (frozen sections) incubated for 9 hours at +4°C. Fixed in formaldehyde, permeabilized using 0.2% Triton X-100. Blocked using 2% BSA for 30 minutes at 20°C. Secondary used at a 1/200 dilution polyclonal Goat anti-mouse IgG conjugated to Alexa Fluor® 555.

    See Abreview

  • ab2868 staining Ryanodine Receptor in Rat cardiomyocyte cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with methanol/acetone (1:1) and blocked with 3% BSA for 1 hour at 18°C. Samples were incubated with primary antibody (1/300 in PBS + 3% BSA) for 16 hours at 4°C. An Alexa Fluor® 546-conjugated Goat anti-mouse IgG (H+L) polyclonal (1/300) was used as the secondary antibody.

    See Abreview

References

This product has been referenced in:
  • Zügel M  et al. Moderate intensity continuous training reverses the detrimental effects of ovariectomy on RyR1 phosphorylation in rat skeletal muscle. Mol Cell Endocrinol 481:1-7 (2019). Read more (PubMed: 30465874) »
  • Estañ MC  et al. Recessive mutations in muscle-specific isoforms of FXR1 cause congenital multi-minicore myopathy. Nat Commun 10:797 (2019). Read more (PubMed: 30770808) »
See all 41 Publications for this product

Customer reviews and Q&As

1-10 of 12 Q&A

Question

1. Order details:
- Batch number:
- Abcam product code: ab6495, ab59225 , ab2868
- Antibody storage conditions (temperature/reconstitution etc)
Store at -20 ℃
2. Please describe the problem (high background, wrong band size, more bands, no band etc).
There is no reaction. I can’t see any bands, even wrong band in the tests.
Moreover, milk with primary antibody were spoiled (color changed) in cool room in last two tests.
I already changed couple ways, however those antibodies didn’t work.

3. On what material are you testing the antibody in WB?
- Species: mice’s hearts
- What’s cell line or tissue
- Cell extract or Nuclear extract:
- Purified protein or Recombinant protein: purified protein
3. The lysate
- How much protein was loaded: 15 µg
- What lysis buffer was used: RIPA
- What protease inhibitors were used:
- What loading buffer was used:
- Phosphatase inhibitors
- Did you heat the samples: temperature and time: 92-95 ℃ , 10 mins
Our samples can be used for other primary antibodies.
4. Electrophoresis/Gel conditions/ Transfer conditions
- Reducing or non reducing gel:
- Reducing agent:
- Gel percentage : ab6495 - 10 %, and 4%-15% (commercial gel)
- ab59225 , ab2868 – 6%
- Transfer conditions: (Type of membrane, Protein transfer verified):
Pure nitrocellulose membrane (pore size 0.2 µm, PROTRAN)
ab6495 - transfer 120 V, 1.5 hours

ab59225 , ab2868 – 6% , wet transferring protein overnight at low voltage, adding 1 % SDS to transfer buffer and reduce metnanol to 10 % or less.
5. Blocking conditions
- Buffer: TBS
- Blocking agent: milk, BSA, serum, what percentage: 5 % milk
- Incubation time: 1 hour
- Incubation temperature: 4 ℃
6. Primary Antibody
- Species
- Reacts against:
- At what dilution(s) have you tested this antibody:
- What dilution buffer was used:
- Incubation time:
- Incubation temperature:
- What washing steps were done:
- ab6495 – mouse, dilution-1/100, incubation overnight, at 4 ℃
cool room
- ab59225- mouse, dilution- 1/1000, incubation overnight, at 4 ℃
cool room
- ab2868- mouse, dilution- 1/2500, incubation overnight, at 4 ℃
cool room
7. Secondary Antibody
- Species:
- Reacts against:
- At what dilution(s) have you tested this antibody:
- Incubation time:
- Wash steps:
- Fluorochrome or enzyme conjugate:
- Do you know whether the problems you are experiencing come from the secondary?
The seconday antibodies are working to our experiments. I don’t think it’s a problem.
8. Detection method
ECl, ECl+, other detection method: ECL
9. Did you apply positive and negative controls along with the samples? Please specify.
10. Optimization attempts
- How many times have you tried the Western? 5 times
- Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control):
- Do you obtain the same results every time e.g. are background bands always in the same place? Almost no bands
- What steps have you altered? Primary antibody concentration, primary antibody incubation time, Transfer time and transfer solution composition (methanol)

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Answer

I am sorry to hear you have been experiencing problems with our antibodies. The quality of our products is important to us and I would like to reassure you that we investigate all customer complaints.

I would like to investigate this particular case further for you, and also obtain more information for our quality records. In order to proceed with this, there are a few things I would like to clarify to better understand and solve the problems experienced with this antibody.

- Could you please send the order details for each antibody? It is important for us to monitor the quality of the antibodies, and also dismiss any problems that may have occurred during the shipping.

- Did all three antibodies give the exact problem? Could you please specify for each antibody what the problem is? If you could please send images of the blots that would be of very much help to understand the problem.

Regarding the blocking with milk, I am afraid it is not recommended as a blocking agent when phospho-proteins, as the casein could break the phosphorilated positions, causing degradations products leading to unspecific bands.

Thank you for your cooperation. I look forward to receiving your reply. Please do not hesitate to contact us if you need any more advice or information.

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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.

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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.

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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 (https://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 (https://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: https://www.abcam.com/index.html?datasheet=119204). Click here (or use the following: https://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 (https://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.

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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.

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Question

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)

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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.

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Question

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE cellular extract from cultured Sprague-Dawley rat hippocampal neurons PRIMARY ANTIBODY 1:1000 dil in 0.5 % blocking buffer in PBS overnight at 4 degees. 4x15 min with PBST prior to secondary. DETECTION METHOD SuperSignal West Pico Chemiluminescent Substrate (pierce) ANTIBODY STORAGE CONDITIONS Aliquoted on receiving in 2 ul aliqots in 0.2 ml tubes, stored at -80 SAMPLE PREPARATION After treatments, cultures are washed, on ice, in PBS. Cells are scraped in Laemlli buffer (4%SDS in tris), samples collected in 1.5 ml tubes and boiled for 15 min. They are then put through a 1 ml syringe with a 27 gauge needle to break up DNA. Samples are spun down in a centrifuge for 15 min at 14,000 rpm, and mixed in a buffer containing glycerol, mercaptoethanol, sds, and hepes (with bromophenol blue). They are frozen at -80 at this point, thawed when required, and heated to 90-100 degrees for 5 minutes prior to loading. AMOUNT OF PROTEIN LOADED 20 micrograms ELECTROPHORESIS/GEL CONDITIONS Reducing conditions, 5-8% tris/acetate gradient gel for 2.5 hours at 150 v TRANSFER AND BLOCKING CONDITIONS Transfer using iBlot imaging system (Invitrogen) http://www.interactivebioscience.cz/admin/_docs/protokol%20-%20Invitrogen%20iBlot%20Western%20Blotting.pdf 4 hour block at RT with 1% blocking buffer (Roche) in PBST (0.05 % tween). SECONDARY ANTIBODY 1:10,000 dil goat anti-mouse antibody (diluted 1:2 in 100% glycerol on receipt) HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3-4 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? I've tried overnight wet transfers, with no noticeable transfer. Using the iBlot system has provided me the best transfer of large proteins (I use a HiMark protein ladder [Invitrogen] with a high protein marker of around 450 kDa, and the highest band is transferred). I've tried different antibodies and ECL kits, and I have never gotten a signal, even though I've read a recent paper showing a group that got a signal with the same amount of protein from the same type of tissue cultures.

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Answer

Thank you for contacting technical support regarding your problem with ab2868. I have tried to find the paper that you mention to see what protocol they have used and unfortunately was unable to find it; if possible could you please send me the reference or PDF so I can look at it? It would be useful to know if the preparation of the cells (age of animals, cell culture prep etc) is the same as your cell culture prep, as it may be that your cells currently have low amounts of Ryanodine Receptor. A good way of determining this would be to run a positive control along your samples, such as rat skeletal muscle lysate (should you not have access to this tissue in your labs, you can purchase it from Abcam, reference ab29376). The following suggestions may also help boost your signal: -try a different lysis buffer to isolate the protein from the membrane: I recommend using RIPA or NP40 buffer with protease inhibitors; I enclose below the procedure: Nonidet-P40 (NP40) buffer 150 mM sodium chloride 1.0% NP-40 (Triton X-100 can be substituted for NP-40) 50 mM Tris, pH 8.0 This is a popular buffer for studying proteins that are cytoplasmic, or membrane-bound, or for whole cell extracts. If there is concern that the protein of interest is not being completely extracted from insoluble material or aggregates, RIPA buffer may be more suitable, as it contains ionic detergents that may more readily bring the proteins into solution. RIPA buffer (Radio Immuno Precipitation Assay buffer) 150 mM sodium chloride 1.0% NP-40 or Triton X-100 0.5% sodium deoxycholate 0.1% SDS (sodium dodecyl sulphate) 50 mM Tris, pH 8.0 RIPA buffer is also useful for whole cell extracts and membrane-bound proteins, and may be preferable to NP-40 or Triton X100-only buffers for extracting nuclear proteins. It will disrupt protein-protein interactions and may therefore be problematic for immunoprecipitations/pull down assays. -blocking: I would recommend using 5% milk as the Roche blocking buffer may not be compatible with the primary antibody and prevent it from detecting the protein. -try more concentrated antibody (diluted in TBST containing Tween20) eg 1:500 We do not recommend to aliquot antibodies in very small volumes as they are not stable in those conditions, it may be that the 2ul aliquots have suffered in the -80C. In the future please try to keep the volumes to 10ul aliquots. I hope these recommendations will help you, please do not hesitate to contact us again if you need further assistance and should the antibody still not work in the positive control with those changes we can offer you a refund or replacement if it was purchased in the last 120 days.

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Thank you for your telephone enquiry yesterday. I am pleased to have obtained some western blot information for you. Ryanodine Receptor antibody [34C] ab2868 has been extensively used in Western blotting in the following references, which should serve as good protocol guides. P. Walton, et al. Ryanodine and Inositol Trisphosphate Receptors Coexist in Avian Cerebellar Purkinje Neurons. J. Cell Biol., 113(5): 1145-1157, 1995. http://www.jcb.org/cgi/content/abstract/113/5/1145 Briefly: "Microsomal samples for SDS-PAGE (16) were adjusted to a protein concentration of 5 mg/ml and mixed 1:1 with 2x concentrated load buffer to give a final SDS concentration of 2%. Samples of solubilized proteins from the sucrose gradient fractions were prepared in a 4x concentrated load buffer. Aliquots of 40-50 ~tl were loaded onto continuous 4-20 % linear polyacrylamide gradient SDS minigels and run under a constant voltage of 130 V at 0-4°C. Gels were stained for protein with 0.2 % Coornassie brilliant blue. Proteins were blotted onto nitrocellulose at an initial setting of 100 V for the first 2 h and then at 40 V overnight in 10 mM 2[N-cyclohexylamino] ethane-suifonic acid, pH 9.6; 10% ethanol (41). Proteins transferred to the nitrocellulose were detected by staining with 0.5% Ponceau S in 5 % acetic acid and destaining in water. Blots were probed with the monoclonal, anti-avian skeletal muscle ryanodine receptor antibody, 34C..." 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 I hope that these are helpful, and please do not hesitate to contact me if you have any other questions.

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Thank your for your enquiry. This product was created against partially purified ryanodine receptor from chicken pectoral muscle. Unfortunately, the exact epitope sequence has not yet been mapped at this time.

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Thank you for your enquiry. The originator of the antibody replied as follows: A general protocol should work well for your customer. RyR is a large protein (~565) and I actually have had individuals have difficulty especially when they are not used to such a target protein. So, I compiled some helpful tips. I have added these tips below for you. In addition, it is really important the customer uses a molecular weight marker that has a high range. I know Amersham carries these. Furthermore, it is important to run suitable controls (i.e. rat skeletal muscle extracts). Please let me know if I may be of further assistance. Below are some helpful tips that the originator has compiled to help optimize high molecular proteins. 1. Add 0.1% (or higher and you may have to play around with this) of SDS to the Transfer Buffer (Tris, glycine, methanol) to increase the transfer efficiency. SDS will aid in eluting the high molecular proteins from the gel, but it can reduce the binding efficiency of these proteins to the nitrocellulose membrane. 2. Alcohol (Methanol) in the transfer buffer improves binding of the proteins to the nitrocellulose only. Elimination of methanol results in increased transfer efficiency but diminishes binding efficiency to the nitrocellulose. 3. The use of a PVDF membrane for protein transfer eliminates the alcohol requirement (a good strategy for high molecular weight proteins). With the use of a PVDF membrane, increases the chance of obtaining information from rare, low abundance proteins. PVDF membranes exhibit better binding efficiency in the presence of SDS in the transfer buffer. PVDF must first be wetted in 100% MeOH but can then be used in buffer which does not contain MeOH. 4. I would recommend, with high molecular weight proteins, to use a wet-tank transfer apparatus. Generally, high molecular proteins should be transferred overnight (~18 hours) at 4 degrees and be sure to place it on a stir plate with a magnetic bar rotating to evenly distribute the buffer as it is transferring. High molecular weight proteins may need to transfer longer than usual and you may even need to play with the voltage (turn it up). 5. If the antigen is highly expressed 20-25 ug may be sufficient. But, if the antigen is low you may have to increase it to 75-100 ug. 6. Use a low% resolving gel or a gradient gel. Also, you can not eliminate methanol, but decrease the %. Most tris-glycine buffers use methanol at 20%, so you could decrease it to 5%.

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