Overview

  • Product name
    Anti-HCN4 antibody [SHG 1E5]
    See all HCN4 primary antibodies
  • Description
    Rat monoclonal [SHG 1E5] to HCN4
  • Host species
    Rat
  • Tested applications
    Suitable for: WB, ICC/IF, IHC-Fr, IHC-FoFr, Flow Cyt, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
    Predicted to work with: Chimpanzee
  • Immunogen

    Synthetic peptide corresponding to Human HCN4 aa 1048-1085.
    Sequence:

    SHGSLLLPPASSPPPPQVPQRRGTPPLTPGRLTQDLKL


    Database link: Q9Y3Q4

  • Positive control
    • IHC-P: Normal and cancer biopsies of human heart tissue. Normal and cancer biopsies of human tonsil tissue. WB: Rat eye lysate. Flow Cytometry: PC-12 cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab32675 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
WB 1/5000. Detects a band of approximately 132 kDa (predicted molecular weight: 130 kDa).
ICC/IF Use at an assay dependent concentration. PubMed: 19775764
IHC-Fr 1/1000.
IHC-FoFr Use at an assay dependent concentration. PubMed: 29403069
Flow Cyt 1/50.

ab18407 - Rat monoclonal IgG1, is suitable for use as an isotype control with this antibody.

IHC-P 1/10 - 1/100.

Target

  • Relevance
    HCN4 is a member of the family of hyperpolarization activated and cyclic nucleotide gated (HCN) channels. HCN currents have been linked to pacemaker activity in the heart and brain, resting potential control, as well as neuronal plasticity. It has been shown that HCN4 channels function as receptors for sour taste, and are associated with pacemaker potential generation in the sinoatrial node.
  • Cellular localization
    Membrane; multi pass membrane protein.
  • Database links
  • Alternative names
    • HCN 4 antibody
    • Hyperpolarization activated cyclic nucleotide gated potassium channel 4 antibody
    • Potassium/sodium hyperpolarization activated cyclic nucleotide gated channel 4 antibody

Images

  • Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized human heart tissue tissues. To expose target proteins heat induced antigen retrieval was performed using 10 mM 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 1:20 with a rat monoclonal antibody recognizing HCN4 ab32675 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.

  • Anti-HCN4 antibody [SHG 1E5] (ab32675) at 1/200 dilution + Rat eye lysate at 25 µg

    Secondary
    HRP Conjugate

    Developed using the ECL technique.

    Predicted band size: 130 kDa
    Observed band size: 132 kDa
    why is the actual band size different from the predicted?

  • Overlay histogram showing PC-12 (Rat adrenal gland pheochromocytoma cell line) cells stained with ab32675 (red line). The cells were fixed with methanol (5 minutes) and then permeabilized with 0.1% PBS-Tween for 20 minutes. 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 (ab32675, 1/50 dilution) for 30 minutes at 22°C. The secondary antibody used was DyLight® 488 goat anti-rat IgG (H+L) (ab98386) at 1/500 dilution for 30 minutes at 22°C. Isotype control antibody (black line) was rat IgG1 [RTK2071] (ab18412, 2 µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a decreased signal in PC-12 cells fixed with 4% paraformaldehyde (10 minutes)/permeabilized with 0.1% PBS-Tween used under the same conditions.

  • Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized human tonsil tissue tissues. To expose target proteins heat induced antigen retrieval was performed using 10 mM 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 1:20 with a rat monoclonal antibody recognizing HCN4 ab32675 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.

References

This product has been referenced in:
  • Li Y  et al. Genetic targeting of Purkinje fibres by Sema3a-CreERT2. Sci Rep 8:2382 (2018). IHC-FoFr ; Mouse . Read more (PubMed: 29403069) »
  • Li Y  et al. Transcription factor TBX18 promotes adult rat bone mesenchymal stem cell differentiation to biological pacemaker cells. Int J Mol Med 41:845-851 (2018). ICC/IF . Read more (PubMed: 29207072) »
See all 19 Publications for this product

Customer reviews and Q&As

1-7 of 7 Abreviews or Q&A

Answer

Thank you for your response.

"No primary control" means that you perform the staining protocol as normal (fixation, blocking, secondary antibody, detection) - except you need to omit the incubation step with the primary antibody. Instead of applying the primary antibody, you only incubate the samples with the dilution buffer you would normally use for diluting the primary antibody.

Isotype control meansyou perform the staining protocol as normal butt the samples will be treated with the isotype control instead of the primary antibody. The isotype of this primary antibody is IgG1, so the isotype control is rat IgG1 .

I would advise you to visit this site to get some further information about positive control and isotype control:

https://www.abcam.com/index.html?pageconfig=technicalfaqs

I hope this helps and if I can assist further, please do not hesitate to contact me.

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Answer

Thank you for your enquiry and your interest in our products.

The immunogen used to raise this antibody was a synthetic peptide: SHGSLLLPPA SSPPPPQVPQ RRGTPPLTPG RLTQDLKL, corresponding to amino acids 1048-1085 of Human HCN4:SHGSLLLPPASSPPPPQVPQRRGTPPLTPGRLTQDLKL. Unfortunately, the peptide is not commercially available.

Even though the immunogen peptide is not available, you could apply several controls to check the specificity of this antibody:

- isotype control,

- negative control,

- no primary - only secondary control

The isotype of this antibody is IgG1 so you can apply an isotype control using rat IgG1 (ab46745, ab128477, ab18403, ab18404, ab18407).

HCN4 is highly expressed in thalamus, testis and in heart, both in ventricle and atrium. It is detected at much lower levels in amygdala, substantia nigra, cerebellum and hippocampus. Generally, negative controls are cells/tissue which do not express the target protein or at very low level. You may find some useful information about expression level of HCN4 at this site: http://www.proteinatlas.org/ENSG00000138622

I hope this helps and if I can assist further, please do not hesitate to contact me.

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Question
Answer

DISCOUNT CODES:
Ab109235: ***
Ab32675: ***

Expiration date: October 5, 2012

Thesecodes will each give you 1 free primary antibody before the expiration date. To redeem this offer, please submit an Abreview for each antibody with rabbit samples and include the correspondingcode in the “Additional Comments” section so we know the Abreview is for this promotion. Please remember that submission of the Abreview is sufficient for the discount code to become active. For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.

Eachcode will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code.

Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

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Answer

Vielen Dank dafür, dass meine Fragen zu beantworten.


Ich möchte darauf kurz hinweisen, dass alle Antikörper eine abgestimmte Optimierung benötigen. Es ist daher nichts Ungewöhnliches ein paar Optimierungsexperimente mit einem neuen Antikörper durchführen zu müssen.


Um Ihr Problem zu lösen, möchte ich die folgenden Vorschläge zu Veränderung Ihres Protokolls machen:

1. Die Antigen Maskierung mit dem Dampfkochtopf ist eine relativ harte Methode. Wir haben gute Ergebnisse mit nur 8 bis 15 min. Mikrowellen Behandlung erzielt. Wenn Sie keine Mikrowelle zur Verfügung haben, reduzieren Sie die Zeit im Dampfkochtopf auf 5 min. Maximum.

2. Da manche Epitope sehr empfindlich auf H2O2 reagieren können, blocken Sie die endogenen Peroxidase nach der primären Antikörper- Inkubation.

Bitte lassen Sie mich 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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Question



) Abcam product code ab 32675 (HCN4)

2) Abcam order reference number or product batch number: LotNr GR75526-1

3) Description of the problem : No staining at all

4) Sample preparation:
Species
Type of sample: PFA/formalin fixed paraffin embedded sections,:
Sample preparation: Formalin fixed paraffin embedded
Positive control human braun and human heart
Negative control: without primaty antibody

5) Fixation step
Yes
If yes: Fixative agent and concentration: 10% buffered Formalin
Fixation time: 5h
Fixation temperature: RT

6) Antigen retrieval method: Tris EDTA pH: 9 and Citrate pH:6

7) Permeabilization method:
Did you do a permeabilization step (details please) or add permeabilizing agent in any dilution buffers? Tried with and without
Permeabilizing agent and concentration: 1% triton-x100 in PBS


8) Blocking agent (eg BSA, serum…): goat serum
Concentration: 2%
Blocking time : 30 min
Blocking temperature : RT

9) Endogenous peroxidases blocked? Yes with methanol and H2O2
Endogenous biotins blocked? No?

10) Primary antibody (If more than one was used, describe in “additional notes”) :
Concentration or dilution : 1:50, 1:100; 1:200; 1:500
Diluent buffer PBS with BSA
Incubation time Over night 4°C

11) Secondary antibody: anti-rat igG biotylinalted (VECTOR)
Species: rabbit
Reacts against: rat IgG
Concentration or dilution : 1:500
Diluent buffer : PBS with BSA
Incubation time 30 min
Fluorochrome or enzyme conjugate: biotynilated (ABC kit HRP-coppeled see below at 13)

12) Washing after primary and secondary antibodies:
Buffer PBS + 0,05% tween
Number of washes 3 times 5 min

13) Detection method
ABC elite kit for peroxidase substrate AND Vector VIP Peroxidase substrate Pink

14) How many times have you run this staining? 3times
Do you obtain the same results every time? Yes
What steps have you altered to try and optimize the use of this antibody?
Different Antibody concentrations 1:50, 1:100; 1:200; 1:500,
tried with and wthout permeabilisation; tried with Alexa-Fluor coppeld secondary antibody (IF)

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Answer

Es tut mir leid, dass Sie Probleme mit unserem Antikörper hatten.

Ich habe da noch ein zwei Fragen bezüglich des Protokolls:

1. Wie lange haben Sie die Antigen Maskierung durchgeführt? Haben Sie eine Zeitreihe durchgeführt?

2. Wann und wie lange haben Sie die endogene Peroxidase geblockt?

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Answer

Thank you very much for your interest in ab32675.

To our knowledge,ab32675 has not been tested in immunoprecipitation (IP). Therefore, I can offer a discount off a future purchase if you buy ab32675 now, test it inIP 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.

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 ab32675 in IP. I will then send a discount code. This code must be issued before purchasing ab32675 so please wait for my reply before ordering.

2. Purchase ab32675 either by phone, fax, or online (www.abcam.com).

3. Test it in IP.

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 how to submit an Abreview, 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 anyprimary antibodyordered 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 ab32675 turns out to be unsuitable for IP, 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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Answer

Thank you for your enquiry. Further to your telephone enquiry I have been in correspondence with the source of this antibody. Unfortunately the immunogen peptide has been exhausted for this antibody and therefore unavailable for purchase. The synthetic peptide used to generate this product corresponds to amino acid residues 1048-1085 of human HCN4 [S(1048) HGSLLLPPASSPPPPQVPQRRGTPPLTPGRLTQDLKL (1085)] Bands that are larger than the expected target protein size could be due to a variety of reasons such as: 1. Post translational modification of the protein; phosphorylated, glycosylated, splicing variant, or due to cross-reactivity with the secondary. 2. Reducing agents can also cause target proteins to be visualized at unexpected sizes. From the details that I have an expected molecular weight of 132kDa is expected. This antibody was applied in the following publication; European Journal of Neuroscience, Volume 17:2084-2096, May 2003. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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