Anti-SERCA2 ATPase antibody [IID8] (ab2817)

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Overview

  • Product name
    Anti-SERCA2 ATPase antibody [IID8]
    See all SERCA2 ATPase primary antibodies
  • Description
    Mouse monoclonal [IID8] to SERCA2 ATPase
  • Host species
    Mouse
  • Specificity
    Detects Sarcoplasmic or Endoplasmic Reticulum Calcium 2 (SERCA2) ATPase. This antibody does not discriminate between the two isoforms. By Western blot, this antibody detects an ~110 kDa protein representing SERCA2 ATPase from canine skeletal muscle triad preparations. Immunofluorescence staining of SECRA2 ATPase in rabbit skeletal muscle results in strong labeling of the entire type I (slow) myofiber consistent with sarcoplasmic reticulum localization. This antibody is not recommended for Western blot detection of rat SERCA2.
  • Tested applications
    Suitable for: ICC/IF, IHC-Fr, WB, ICC, Inhibition Assay, ELISA, IP, IHC-P, Flow Cytmore details
  • Species reactivity
    Reacts with: Rat, Sheep, Rabbit, Guinea pig, Hamster, Cow, Dog, Human, Pig
    Predicted to work with: Non human primates, Amphibian
  • Immunogen

    Full length native protein (purified) corresponding to Dog SERCA2 ATPase. Purified canine cardiac sarcoplasmic reticulum.

  • Positive control
    • canine skeletal muscle triad preparations

Properties

Applications

Our Abpromise guarantee covers the use of ab2817 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 at an assay dependent concentration.
IHC-Fr 1/500.
WB 1/2500. Detects a band of approximately 100 kDa.
ICC Use at an assay dependent concentration.
Inhibition Assay Use at an assay dependent concentration.
ELISA Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
IHC-P Use a concentration of 1 µg/ml.
Flow Cyt Use 1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

Target

  • Function
    This magnesium-dependent enzyme catalyzes the hydrolysis of ATP coupled with the translocation of calcium from the cytosol to the sarcoplasmic reticulum lumen. Isoform 2 is involved in the regulation of the contraction/relaxation cycle.
  • Tissue specificity
    Isoform 1 is widely expressed in smooth muscle and nonmuscle tissues such as in adult skin epidermis, with highest expression in liver, pancreas and lung, and intermediate expression in brain, kidney and placenta. Also expressed at lower levels in heart and skeletal muscle. Isoforms 2 and 3 are highly expressed in the heart and slow twitch skeletal muscle. Expression of isoform 3 is predominantly restricted to cardiomyocytes and in close proximity to the sarcolemma. Both isoforms are mildly expressed in lung, kidney, liver, pancreas and placenta. Expression of isoform 3 is amplified during monocytic differentiation and also observed in the fetal heart.
  • Involvement in disease
    Defects in ATP2A2 are a cause of acrokeratosis verruciformis (AKV) [MIM:101900]; also known as Hopf disease. AKV is a localized disorder of keratinization, which is inherited as an autosomal dominant trait. Its onset is early in life with multiple flat-topped, flesh-colored papules on the hands and feet, punctate keratoses on the palms and soles, with varying degrees of nail involvement. The histopathology shows a distinctive pattern of epidermal features with hyperkeratosis, hypergranulosis, and acanthosis together with papillomatosis. These changes are frequently associated with circumscribed elevations of the epidermis that are said to resemble church spires. There are no features of dyskeratosis or acantholysis, the typical findings in lesions of Darier disease.
    Defects in ATP2A2 are the cause of Darier disease (DD) [MIM:124200]; also known as Darier-White disease (DAR). DD is an autosomal dominantly inherited skin disorder characterized by loss of adhesion between epidermal cells (acantholysis) and abnormal keratinization. Patients with mild disease may have no more than a few scattered keratotic papules or subtle nail changes, whereas those with severe disease are handicapped by widespread malodorous keratotic plaques. In a few families, neuropsychiatric abnormalities such as mild mental retardation, schizophrenia, bipolar disorder and epilepsy have been reported. Stress, UV exposure, heat, sweat, friction, and oral contraception exacerbate disease symptoms. Prevalence has been estimated at 1 in 50000. Clinical variants of DD include hypertrophic, vesicobullous, hypopigmented, cornifying, zosteriform or linear, acute and comedonal subtypes. Comedonal Darier disease (CDD) is characterized by the coexistence of acne-like comedonal lesions with typical Darier hyperkeratotic papules on light-exposed areas. At histopathologic level, CDD differs from classic DD in the prominent follicular involvement and the presence of greatly elongated dermal villi.
  • Sequence similarities
    Belongs to the cation transport ATPase (P-type) (TC 3.A.3) family. Type IIA subfamily.
  • Post-translational
    modifications
    Nitrated under oxidative stress. Nitration on the two tyrosine residues inhibits catalytic activity.
  • Cellular localization
    Endoplasmic reticulum membrane. Sarcoplasmic reticulum membrane.
  • Information by UniProt
  • Database links
    see all
  • Alternative names
    • AT2A2_HUMAN antibody
    • Atp2a2 antibody
    • ATP2B antibody
    • ATPase Ca++ transporting cardiac muscle slow twitch 2 antibody
    • Calcium pump 2 antibody
    • Calcium-transporting ATPase sarcoplasmic reticulum type antibody
    • Calcium-transporting ATPase sarcoplasmic reticulum type slow twitch skeletal muscle isoform antibody
    • Cardiac Ca2+ ATPase antibody
    • DAR antibody
    • DD antibody
    • Endoplasmic reticulum class 1/2 Ca(2+) ATPase antibody
    • MGC45367 antibody
    • Sarcoplasmic/endoplasmic reticulum calcium ATPase 2 antibody
    • SERCA 2 antibody
    • SERCA2 antibody
    • serca2a antibody
    • slow twitch skeletal muscle isoform antibody
    • SR Ca(2+)-ATPase 2 antibody
    see all

Images

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SERCA2 ATPase antibody [IID8] (ab2817)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SERCA2 ATPase antibody [IID8] (ab2817)
    ab28217 (2µg/ml) staining calpain S1 in renal medulla
    Sections were stained using an automated system (DAKO Autostainer Plus ), at room temperature: sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffers EDTA pH 9.0 . Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SERCA2 ATPase antibody [IID8] (ab2817)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SERCA2 ATPase antibody [IID8] (ab2817)
    ab2817 (1µg/ml) staining SERCA2 ATPase in human heart (left ventricle), using an automated system (DAKO Autostainer Plus). Using this protocol there is strong cytoplasmic staining of cardiomyocytes.
    Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH6.1 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.
  • Immunocytochemistry/ Immunofluorescence - Anti-SERCA2 ATPase antibody [IID8] (ab2817)
    Immunocytochemistry/ Immunofluorescence - Anti-SERCA2 ATPase antibody [IID8] (ab2817)
    ICC/IF image of ab2817 stained MCF7 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 (ab2817, 5µg/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.

  • Western blot - Anti-SERCA2 ATPase antibody [IID8] (ab2817)
    Western blot - Anti-SERCA2 ATPase antibody [IID8] (ab2817)
    All lanes : Anti-SERCA2 ATPase antibody [IID8] (ab2817) at 1 µg/ml

    Lane 1 : Human skeletal muscle tissue lysate - total protein (ab29330)
    Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 3 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Observed band size: 99 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 30 kDa, 44 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 8 minutes


    The 99 kDa band observed is comparable to the molecular weight seen with other commercially available antibodies to SERCA2 ATPase.
  • Flow Cytometry - Anti-SERCA2 ATPase antibody [IID8] (ab2817)
    Flow Cytometry - Anti-SERCA2 ATPase antibody [IID8] (ab2817)
    Overlay histogram showing HepG2 cells stained with ab2817 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab2817, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SERCA2 ATPase antibody [IID8] (ab2817)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SERCA2 ATPase antibody [IID8] (ab2817)
    Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized human heart tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH 6.0) 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 1/200 with a mouse monoclonal antibody recognizing SERCA2 ATPase (ab2817) 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.
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SERCA2 ATPase antibody [IID8] (ab2817)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SERCA2 ATPase antibody [IID8] (ab2817)
    Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized human liver 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 1/200 with a mouse monoclonal antibody recognizing SERCA2 ATPase (ab2817) 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.
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SERCA2 ATPase antibody [IID8] (ab2817)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SERCA2 ATPase antibody [IID8] (ab2817)
    Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized human tonsil tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH 6.0) 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 1/200 with a mouse monoclonal antibody recognizing SERCA2 ATPase (ab2817) 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.
  • Immunocytochemistry/ Immunofluorescence - Anti-SERCA2 ATPase antibody [IID8] (ab2817)
    Immunocytochemistry/ Immunofluorescence - Anti-SERCA2 ATPase antibody [IID8] (ab2817)
    Immunofluorescent analysis of SERCA2 ATPase using SERCA2 ATPase Monoclonal antibody (IID8) ab2817 shows staining in U251 glioma cells. SERCA2 ATPase staining (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing SERCA2 ATPase ab2817 at a dilution of 1:100-1:200 over night at 4 ?C washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

  • Immunocytochemistry/ Immunofluorescence - Anti-SERCA2 ATPase antibody [IID8] (ab2817)
    Immunocytochemistry/ Immunofluorescence - Anti-SERCA2 ATPase antibody [IID8] (ab2817)
    Immunofluorescent analysis of SERCA2 ATPase using SERCA2 ATPase Monoclonal antibody (IID8) ab2817 shows staining in A549 cells. SERCA2 ATPase staining (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing SERCA2 ATPase ab2817 at a dilution of 1:100-1:200 over night at 4 ?C washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

  • Immunocytochemistry/ Immunofluorescence - Anti-SERCA2 ATPase antibody [IID8] (ab2817)
    Immunocytochemistry/ Immunofluorescence - Anti-SERCA2 ATPase antibody [IID8] (ab2817)
    Immunofluorescent analysis of SERCA2 ATPase using SERCA2 ATPase Monoclonal antibody (IID8) ab2817 shows staining in HeLa cells. SERCA2 ATPase staining (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing SERCA2 ATPase ab2817 at a dilution of 1:100-1:200 over night at 4 ?C washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

References

This product has been referenced in:
  • Cao Q  et al. Dickkopf-3 upregulation mediates the cardioprotective effects of curcumin on chronic heart failure. Mol Med Rep 17:7249-7257 (2018). Read more (PubMed: 29568962) »
  • da Silva JS  et al. Adenosine A2A receptor agonist prevents cardiac remodeling and dysfunction in spontaneously hypertensive male rats after myocardial infarction. Drug Des Devel Ther 11:553-562 (2017). IHC-P ; Rat . Read more (PubMed: 28293100) »
See all 23 Publications for this product

Publishing research using ab2817? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-7 of 7 Abreviews or Q&A

Question

phone call: no good signal in mouse and human tissue

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Answer

Selon nos informations vous avez connu quelques difficultés avec ab2817 et avez contacté notre service scientifique.

Après étude de notre correspondance, nous remarquons que nous sommes toujours en attente du questionnaire qui nous permettrait de vous aider à résoudre ce problème. Si vous avez déjà envoyé ce questionnaire, il semble que celui-ci n’est pas était pris en compte par notre système et je vous serais gré de me le transmettre de nouveau. Veuillez nous excuser par le désagrément causé par cette mesure.

Dans le cas contraire, pourriez-vous me faire savoir si vous avez déjà résolu ce problème et ainsi nous pourrions clore ce dossier dans notre système.

Je vous souhaite bonne chance dans vos recherches. N’hésitez pas à me recontacter pour plus d’assistance.

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Question

phone call: no good signal in mouse and human tissue

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Answer

Je suis désolée d'apprendre que ab2817 ne vous donne pas de bons résultats en Western blot sur du tissu humain ou murin.

Veuillez trouver ci-joint un questionnaire discuté au téléphone qui nous permet de regrouper le plus d'informations possible sur le protocole que vous avez utilisé avec cet anticorps.
Celui-ci nous permettra de vous fournir la meilleure assistance technique.

Si nous concluons que notre produit est fautif et ne fonctionne pas comme explicité par notre fiche technique, nous serons ravis de mettre en place l'envoi d'un tube de remplacement gratuit ou d'un remboursement (sous réserve d'une commande ultérieure de 180 jours).

Je vous remercie par avance de nous envoyer votre protocole.

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Question

Hello, we recently ordered the anti-SERCA2 mouse monoclonal antibody (AB2817) and i found that the results obtained were not as good as with a previous batch of that antibody (under identical conditions). Is there anything we can do to rectify this situation? Best wishes

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Answer

Thank you for contacting us. I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly. I am attaching our questionnaire so that we can gather further information regarding the samples tested and the protocol used. Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results. This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody. I look forward to receiving your reply.  

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Question

Thank you so much about info of your antibodies, it helped me a lot. I did the scanning for sequence alignments across different species and it seems that ab77289 sequence is quite conserved. In danio there was difference just between 1-2 amino acids, same for tilapia (those are the only two fishes where SERCA2 has been sequenced or sequence has been published). And comparison to frogs, lizards, birds and mammals was even more compatible. And what was nice to notice, in invertebrates the difference was not too bad either. So I think if any of your antibodies are going to work it is the ab77289. Obviously, as you said, there is no certainty that it will work with salmonids but at least I can test it. Thank you Kate! Cheers,

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Answer

Thank you for your reply. From the information you have provided, the alignment with the Danio sounds high, and although we cannot guarantee it without laboratory testing, we would suggest the antibody should detect in this fish. I can recommend to consider submitting an Abreview with the results via the online product datasheet. We always encourage customers to send their results back to us, whether positive or negative, and we make all product information available to researchers. We provide Abpoint rewards for submission of all Abreviews. To find out more about our Abreview system, please see the following webpage: https://www.abcam.com/abreviews I wish you the best of luck with your experiments and research. I hope this has been helpful to you. If you have any questions, please do not hesitate to contact us.

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Question

BATCH NUMBER 194792 ORDER NUMBER 168017 DESCRIPTION OF THE PROBLEM None specific bands SAMPLE Human/Panc-1 cell extract/ PRIMARY ANTIBODY Mouse monoclonal [IID8] to SERCA2 ATPase/in TBST with 5% milk/1:1000/incubate overnight, cold room/ wash : with TBST buffer, 10min x 3times DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED No ANTIBODY STORAGE CONDITIONS -20 SAMPLE PREPARATION High salt lysis buffer/Yes, purchased from sigma/5min boiling AMOUNT OF PROTEIN LOADED 100ug/lane ELECTROPHORESIS/GEL CONDITIONS Non-reducing gel/8% TRANSFER AND BLOCKING CONDITIONS Transfer Buffer 3.5L Tris-Base 10.6g Glycine 10.5g Methanol 350ml / Transfer 4hours, 0.4A /Blocking agent: 5% Non-fat milk SECONDARY ANTIBODY [another company]/Goat anti Mouse IgG1/TBST with 5% milk/1:5000/3hours incubation/ 10min x 3times wash HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

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Answer

Thank you for your enquiry. I am sorry to hear that you have been having difficulties with this antibody. Thank you for taking the time to complete our technical questionnaire. Our mouse monoclonal [IID8] to SERCA2 ATPase (ab2817) has been applied in many publications and we have received relatively few complaints about it. At this stage I would like to make the following recommendations: We recommend that the antibody is applied by western blot at a dilution of 1/2500. Therefore in order to improve the specificity of the antibody I would like to recommend that you reduce the dilution that you have been applying this antibody. This often serves to reduce the intensity of extraneous bands. In order to further reduce non specific bands I would like to recommend that you also reduce the mass of protein that you have been loading from 100ug to ~30ug of extract. This should be more than sufficient to detect this protein. I would also like to suggest that you change the blocking agent from milk to 3% BSA and supplement your antibody incubations with this agent. We often find that this serves to reduce background and/or extraneous bands that may be present. Please get back in touch with me should this not serve to improve your results.

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Question

I need a SERCA2 antibody (which recognizes SERCA2B) for Xenopus laevis. None of your epitopes are available so I cannot evaluate your selection. I have attatched to this e-mail the probable amino acid sequence for this protein in Xenopus and am interested to hear your thoughts on potential antibody choices.

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Answer

Thank you for your enquiry. Regarding ab2817 and ab2861: These monoclonal antibodies have not been epitope mapped, therefore the epitope could be anywhere along the full length of the protein. I did an alignment of Human SERCA2 with the Xenopus SERCA2 sequence you attached to your email, and they are 92% homologous. I am not sure if this information helps you in your decision, but I thought I would let you know. I am still looking into ab3625 to see if I can uncover more information about the peptide that was used to produce this antibody. Once I find anything more, I will let you know. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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Question

DESCRIPTION OF THE PROBLEM I am not getting any bands. For the NCX and SERCA antibodies I am getting a dark smear corresponding to the stacking gel that is not present with the RyR or Alpha2, or with ab3516 and 2865 (these two are working well). SAMPLE The protein is from sheep. I have a total protein prep that is working well for phospholamban, calsequestrin, phosph-ERK and ERK2 detection. I also have a membrane protein prep that I'm not 100% confident with- so far I haven't detected any specific bands by blotting. However, bands from this prep are visible when I do a Coomassie stain. PRIMARY ANTIBODY I am using your primaries at the recommended dilution. I incubated the primaries overnight at 4C. I wash once for 20 minutes with TBST, then rinse again briefly with TBST. DETECTION METHOD I am using Pierce's SuperSignal. I have left the blots to expose for up to 15 minutes, or until the background made it impossible to see anything. Typically I have low background. POSITIVE AND NEGATIVE CONTROLS USED None. ANTIBODY STORAGE CONDITIONS Also antibodies 6495, 2868 and 2864. Antibodies aliquoted and stored at -20. NCX antibody reconstituted in 200ul PBS, aliquoted and stored at -20. SAMPLE PREPARATION Prior to loading on a gel the proteins are mixed with Laemmli buffer. I've tried increasing the ratio of the buffer to the sample. AMOUNT OF PROTEIN LOADED I've tried these with 20ug of protein up to more than a mg. ELECTROPHORESIS/GEL CONDITIONS Standard SDS-PAGE. I've tried running a 4% stacking gel & 12% resolving gel, also a 3% stacking gel and 5% resolving gel. I am now using a small gel system, but I have tried a larger format in the past. TRANSFER AND BLOCKING CONDITIONS The transfer buffer is Tris based and has glycine. I have tried transfering for 30min and 60min on Bio-Rad's Transblot SD. I block with 5% nonfat milk in TBST. SECONDARY ANTIBODY I am using Amersham ECL anti-rabbit (NA934) and anti-mouse (NA931) secondaries at 1:4000, this works extremely well for other primaries in this lab. I wash for 20 minutes with TBST, then rinse again briefly with TBST. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? >3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? No WHAT STEPS HAVE YOU ALTERED? I have tried two different protein extractions. I have changed the proportion of Laemmli loading buffer. I have reduced the percentage of the gel. I have increased the transfer time. ADDITIONAL NOTES I'm hesitant to simply increase the concentration of the primary in the NCX and SERCA preps, in part because there are dark smears in the stacking gel areas of these blots. Also, the NCX and alpha2 antibodies are already used at a high concentration and I could easily use them up trying to get these protocols to work. I would be pleased to immediately try any recommendations you have, however.

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Answer

I'm sorry to hear you are having problems with ab2817 and ab6495. Your detailed protocol was very useful to understand the source of the problem and it is clear your protocol and modifications were very thorough. My suspicions therefore lied with your samples, they are sheep derived and neither antibody has been tested in sheep. I therefore I checked the homology between the immunogen of ab2827 (canine protein) and the sheep protein and I found only 40% homology (119 aa out of 295aa), so I suspect that the antibody ab2827 is not detecting the protein. For ab6495, the homology between the rabbit NCX1 protein and the sheep NCX1 protein was so low that I couldn't find it when BLASTing with the rabbit protein the sheep genome. So I'm sorry to say that it seems that neither antibodies seem to work due to the nature of the samples (on the protocol aspect, ab6495 was tested at a 1:200 dilution with using 5S-protein A; with ECL dilutions may need to be considerably higher regardless of the samples). Please let me know if I can be of further assistance,

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