Overview

  • Product name
    Anti-IP3 receptor antibody
    See all IP3 receptor primary antibodies
  • Description
    Rabbit polyclonal to IP3 receptor
  • Host species
    Rabbit
  • Tested applications
    Suitable for: IP, WB, ICC/IF, ICC, IHC-Fr, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Hamster, Dog, Human
    Predicted to work with: Cow
  • Immunogen

    Synthetic peptide corresponding to Human IP3 receptor aa 1829-1848.
    Sequence:

    NKKKDDEVDRDAPSRKKAKE


    (Peptide available as ab5839)

Properties

Applications

Our Abpromise guarantee covers the use of ab5804 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
IP Use at an assay dependent concentration.
WB 1/1000. Predicted molecular weight: 314 kDa.Can be blocked with IP3 receptor peptide (ab5839).
ICC/IF Use at an assay dependent concentration.
ICC Use at an assay dependent concentration.
IHC-Fr 1/250.

Staining of IP3R-I in rat cerebellum with ab5804 results in staining of Purkinje cells.

IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Target

  • Function
    Intracellular channel that mediates calcium release from the endoplasmic reticulum following stimulation by inositol 1,4,5-trisphosphate.
  • Tissue specificity
    Widely expressed.
  • Involvement in disease
    Defects in ITPR1 are the cause of spinocerebellar ataxia type 15 (SCA15) (SCA15) [MIM:606658]. Spinocerebellar ataxia is a clinically and genetically heterogeneous group of cerebellar disorders. Patients show progressive incoordination of gait and often poor coordination of hands, speech and eye movements, due to degeneration of the cerebellum with variable involvement of the brainstem and spinal cord. SCA15 is an autosomal dominant cerebellar ataxia (ADCA). It is very slow progressing form with a wide range of onset, ranging from childhood to adult. Most patients remain ambulatory.
  • Sequence similarities
    Belongs to the InsP3 receptor family.
    Contains 5 MIR domains.
  • Domain
    The receptor contains a calcium channel in its C-terminal extremity. Its large N-terminal cytoplasmic region has the ligand-binding site in the N-terminus and modulatory sites in the middle portion immediately upstream of the channel region.
  • Post-translational
    modifications
    Phosphorylated by cAMP kinase. Phosphorylation prevents the ligand-induced opening of the calcium channels.
    Phosphorylated on tyrosine residues.
  • Cellular localization
    Endoplasmic reticulum membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • 4 antibody
    • 5-trisphosphate receptor antibody
    • 5-trisphosphate receptor type 1 antibody
    • DKFZp313E1334 antibody
    • DKFZp313N1434 antibody
    • inositol 1 4 5 triphosphate receptor type 1 antibody
    • Inositol 1 4 5 trisphosphate Receptor Type 1 antibody
    • Inositol 1 antibody
    • InsP3R1 antibody
    • IP3 antibody
    • IP3 receptor antibody
    • IP3 receptor isoform 1 antibody
    • IP3R 1 antibody
    • IP3R antibody
    • IP3R1 antibody
    • ITPR 1 antibody
    • Itpr1 antibody
    • ITPR1_HUMAN antibody
    • SCA15 antibody
    • SCA16 antibody
    • SCA29 antibody
    • Type 1 inositol 1 4 5 trisphosphate receptor antibody
    • Type 1 inositol 1 antibody
    • Type 1 InsP3 receptor antibody
    see all

Images

  • Shows immunolocalization of ITPR1 in rat cerebellum using ab5804.

  • IHC image of ab5804 staining in normal human hippocampus formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab5804, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

References

This product has been referenced in:
  • Ausman J  et al. Ceramide-induced BOK promotes mitochondrial fission in preeclampsia. Cell Death Dis 9:298 (2018). Read more (PubMed: 29463805) »
  • Zhao X  et al. Calcium-sensing receptor-mediated L-tryptophan-induced secretion of cholecystokinin and glucose-dependent insulinotropic peptide in swine duodenum. J Vet Sci 19:179-187 (2018). Read more (PubMed: 29284209) »
See all 20 Publications for this product

Customer reviews and Q&As

1-9 of 9 Abreviews or Q&A

Application
Western blot
Sample
Mouse Tissue lysate - whole (Hippocampal)
Gel Running Conditions
Reduced Denaturing (8%)
Loading amount
20 µg
Specification
Hippocampal
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 4% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Jul 13 2018

Application
IHC - Wholemount
Sample
Mouse Tissue (Brain (cerebellum))
Specification
Brain (cerebellum)

Mr. Gabriel Luna

Verified customer

Submitted Jan 05 2018

Answer

Thank you for confirming these details and for your cooperation. As agreed, I have issued a free of charge replacement with the order number ******. To check the status of the order please contact our Customer Service team and reference this number. Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know. I wish you the best of luck with your research.  

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Question

Thank you for your previous guidance (see attached emails below). 1) ab5804 : For explanantions about the extra bands, I had already added sufficient amounts of protease inhibitors in the sample buffer but have since tried making fresh samples with greater amounts and still I am obtaining extra bands. I have also tried a secondary antibody only control and have tried blocking in a range of products. Hopefully the band observed >250kDa is ITPR1 but I feel I will need to use a blocking peptide to confirm this. Also, as I am receiving other bands, I would like to see if they are non-specifics. I am certain that when I looked at the datasheet for this antibody last week there was a blocking peptide associated with it but I notice that the link is no longer available. Is it this product: https://www.abcam.com/ITPR1-peptide-ab5839.html ? I was wondering would there be any way to receive a small sample of this product to test this out? 2) ab77743 : As for this antibody, I have tried all your suggestions and a range of tests but still I am not receiving the 138kDa band. I have now decided it is best to just move on to a different antibody. I see for Western Blots you have 2 further options. https://www.abcam.com/Phospholipase-C-beta-1-antibody-ab21824.html ab21284 when BLASTed shows that there is 100% coverage with bovine but also there is 96% coverage with PLCB3. Unfortunately I need it to be PLB1 specific. I am curious as to why the human and rat sequences also have the 96% coverage. PLCB3 is of the same MW as PLCB1. Does that mean this antibody could possibly be probing for both? https://www.abcam.com/Phospholipase-C-beta-1-antibody-ab103278.html This antibody states it is a Synthetic peptide conjugated to KLH, corresponding to a region within C terminal amino acids 1155-1184 of Human Phospholipase C beta 1. Can I check is this amino acids 1155-1184 of Human Phospholipase C beta 1 isoform A? If so can you please confirm that I am BLASTing the correct sequence - krlple ilefvqeamk gkisedsnhg sapl When I BLAST this the coverage with bovine PLCB1 is 96%. It does however, match lots of other things in bovine but to a much smaller % (40% or less). Can I check with you is this antibody in a 400ul aliquot? Many thanks once again for your time. I look forward to your reply. Best wishes,

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Answer

Thank you for confirming these details and for your cooperation. I can confirm that : - the blocking peptide for ab5804 is ab5839 - the immunogen of ab103278 is aa 1155-1184 of the isoform A (SwissProt: Q9NQ66-1) Because we carry over 70,000 products, it isn't feasible for us to keep small sample sizes of our products. I am happy to issue a free of charge unit of each the above products. In order to prevent any shipping error, could you please send me the original order number(s), the date(s) when the order(s) was(were) placed or at least the shipping address? Thank you.

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Question

Another antibody I have been probing for is the PLCB1 antibody, https://www.abcam.com/Phospholipase-C-beta-1-antibody-ab77743.html. The expected band size is 138kDa. I haven't received a band at this size - please see attached document. The first three lanes after the ladder are in HEK293T lysate and the next 3 lanes are in bovine retinal lysate. The last 2 lanes are HELA lysate and a negative control respectively, followed by the ladder. As you can see, in my lysate of interest -bovine lysates, there are some very strong bands appearing around 55, 45 and some weaker ones ~35kDa. I was hoping to hear any opinions from you before I try to plan my next run so I can consider how to troubleshoot for this problem. On looking at your troubleshooting guide I have decided to make a few changes - to increase the secondary antibody concentration. I will not increase the primary antibody concentrtion as I am already using a 1/500 dilution which is a lot of my antibody and I incubated for 18hours at 4degrees. I loaded 40, 30 and 20ug of lysate for each of the HEK and bovine lysates. I feel 40ug is quite a lot for it not to show anything and so I'm not so sure increasing the amount will fix things, especially as the bands I am seeing ~55 are already very strong. I will also consider that I have washed the membrane for too long and will reduce my %milk with my antibody to 0.5%. The only other thing I was wondering about is that I notice you do not have any references for this antibody and the picture is from Immunoprecipitation. I it that the protein is lowly expressed and I need to use IP to enhance my signal? One other suggestion has been that my sample could be degraded and so the addition of the 35,45 and 55kDa bands sums up to 138kDa of interest. Could this be correct? If so, would it be possible that that will happen to only some of the proteins in the membrane as since developping this blot I have stripped and reprobed for a different protein (a much larger prtoein) which didn't show any signs of degradation. If degradation is a possible explanation, can you suggest how I can prevent this? These samples were very fresh and obtained plenty of protease inhibitors. I hope you can appreciate my million questions in advance of my next experiment as I have very little of this antibody (due to the high concentration used) and so want to consider all my options before wasting it. I would be grateful for any advice/tips/opinions. Many thanks for your help in advance and I look forward to your reply.

Read More
Answer

Thank you for contacting us and for sending images of the results obtained with ab5804 and ab77743. 1)  ab5804 : I looked at the image of the blot and I am positive that the band observed above the 250kDa marker is ITPR1. As explained in my previous email, Western blotting big proteins of more than 300 kDa is not that easy. From your blot, I can see that the transfer went well. Regarding the size, it looks close enough to 300kDa to confirm you targeted the right protein. The values for the expected as well as the observed MW stated on the product datasheet were a typo and the datasheet has been updated. The expected MW for the Human ITPR1 isoform 1 is 314 kDa (http://www.uniprot.org/uniprot/Q14643). The observed molecular weight should fluctuate around this value but should not be, I think, as low as 240 kDa. In general, the apparent molecular weight is affected by the pH/polarity of the buffer giving a different apparent molecular weight in different buffers. The bands observed around 100 kDa may be due to degradation of ITPR1, if so make sure you add protease inhibitors in the sample buffer. It also may be due to some non-specific binding of the antibody to other protein presenting a sequence similar to the immunogen peptide. It may also be due to some non-specific binding of the secondary antibody, to determine this last possibility it is recommended to run a "no primary" control. Finally, I would suggest to try a different blocking agent. 5% BSA can sometimes reduce the background as well as enhance the specific binding. 2) ab77743 : the recommended dilution is 1/200, have you tried the antibody in this condition? An incubation overnight at 4°C is common. You may also try at room temperature for 3 hours. I would recommend not to use milk as blocking agent for phospho-proteins. 5% BSA is recommended.   I hope these suggestions are helpful. If not, do not hesitate to contact us again and to visit our protocols page at  www.abcam.com/protocols and our protocol book available at : https://www.abcam.com/index.html?pageconfig=resource&rid=13111  

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Answer

Thank you very much for your inquiry and your interest in our antibodies. Indeed, we do not have ITPR1 antibodies tested against bovine in our catalogue currently. We are however able to provide you with a testing offer, please see below. In regards to the alignments, I do not have the immunogen sequence of the ab108517 as it is proprietary information of the laboratory from whom we source this antibody. I have however requested the alignment information from them. Please see here below also the alignment of the immunogen sequence of ab97823 with the bovine sequence. The alignment is very good with 93% identity. The alignment of the immunogen of the ab5804 has however 100% identity with the bovine protein, so crossreaction is even more likely. In regards to the blast, I am sorry that I do not really understand the question. I have blasted the immunogen against the human proteins (to have a better overview) and have found only significant reaction with the ITPR1 protein. There are different isoforms, however all from the ITPR1. Please provide me maybe with the relevant sequences. We can provide you also with a testing offer for testing an ITPR1 antibody against bovine. Indeed, even if the immunogen sequence is identical, we do need experimental evidence to be able to guarantee it. Therefore as to our knowledge, ab97823 or ab5804 have not been tested in bovine, I can offer a discount off a future purchase if you buy ab97823 or ab5804 now, test it in bovine 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. This offer might also be applicable to the ab108517, we need however to wait for the alignment results in order to be able to decide on this. If you are interested in this offer, please follow these steps: 1. Reply to this e-mail to let me know that you would like to proceed and test ab97823 or ab5804 in bovine. I will then send a discount code. This code must be issued before purchasing ab97823 or ab5804 so please wait for my reply before ordering. 2. Purchase ab97823 or ab5804 either by phone, fax, or online (www.abcam.com). 3. Test it in bovine. 4. Let us know the results, positive or negative, using our Abreview system (this will take about 10 minutes and images are great if you have them!). To find out more about our Abreview system, please visit: https://www.abcam.com/abreviews. 5. After the review is submitted to us, the discount code becomes active. Simply place your new order by phone, fax, or on the web and mention the discount code. The discount can be redeemed for any primary antibody ordered and the discount code is valid for 4 months after issue. We are always pleased to obtain feedback about our products and any information is greatly appreciated! Even if ab97823 or ab5804 turns out to be unsuitable for bovine, you will still receive the discount on your next purchase after your Abreview has been submitted. Please let me know if you have any questions about this offer and I would be happy to help you further. The Terms and Conditions of this offer can be found at: www.abcam.com/collaborationdiscount.

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunoprecipitation
Sample
Mouse Cell lysate - other (DO11.10 Thymocyte hybridoma)
Total protein in input
25 µg
Specification
DO11.10 Thymocyte hybridoma
Immuno-precipitation step
Protein A/G

Dr. Jonathan Rud

Verified customer

Submitted Dec 28 2009

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (DO11.10 T-cell Hybridoma)
Loading amount
25 µg
Specification
DO11.10 T-cell Hybridoma
Gel Running Conditions
Reduced Denaturing (4% gel)
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Dr. Jonathan Rud

Verified customer

Submitted Dec 28 2009

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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