製品の概要

  • 製品名
    Anti-5HT2A Receptor antibody
    5HT2A Receptor 一次抗体 製品一覧
  • 製品の詳細
    Rabbit polyclonal to 5HT2A Receptor
  • 由来種
    Rabbit
  • アプリケーション
    適用あり: IHC-FoFr, WBmore details
  • 種交差性
    交差種: Mouse, Rat, Human
  • 免疫原

    Synthetic peptide:

    GDGPRLYHNDFNSRDANTSE

    , corresponding to N terminal amino acids 22-41 of Rat 5HT2A Receptor

  • ポジティブ・コントロール
    • Rat cortex, amygdala and hippocampus tissue; mouse frontal cortical neuron cultures.
  • 特記事項
    Upon delivery dilute with phosphate buffer or Tris buffer at dilutions no higher than 1/10, aliquot and freeze at -15° C or lower. Antibody can be stored for up to six months if handled as described above.

製品の特性

アプリケーション

Our Abpromise guarantee covers the use of ab66049 in the following tested applications.

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

アプリケーション Abreviews 特記事項
IHC-FoFr 1/300 - 1/500. Biotin/Streptavidin-HRP Technique.
WB 1/100. Detects a band of approximately 53 kDa (predicted molecular weight: 53 kDa).

ターゲット情報

  • 機能
    This is one of the several different receptors for 5-hydroxytryptamine (serotonin), a biogenic hormone that functions as a neurotransmitter, a hormone, and a mitogen. This receptor mediates its action by association with G proteins that activate a phosphatidylinositol-calcium second messenger system. This receptor is involved in tracheal smooth muscle contraction, bronchoconstriction, and control of aldosterone production.
  • 配列類似性
    Belongs to the G-protein coupled receptor 1 family.
  • ドメイン
    The PDZ domain-binding motif is involved in the interaction with INADL, CASK, APBA1, DLG1 and DLG4.
  • 細胞内局在
    Cell membrane. Localizes to the post-synaptic thickening of axo-dendritic synapses.
  • Information by UniProt
  • 参照データベース
  • 別名
    • 5 HT 2 antibody
    • 5 HT 2A antibody
    • 5 HT2 receptor antibody
    • 5 HT2A antibody
    • 5 hydroxytryptamine receptor 2A antibody
    • 5-HT-2 antibody
    • 5-HT-2A antibody
    • 5-hydroxytryptamine (serotonin) receptor 2A, G protein-coupled antibody
    • 5-hydroxytryptamine 2A receptor antibody
    • 5-hydroxytryptamine receptor 2A antibody
    • 5HT2A_HUMAN antibody
    • HTR 2 antibody
    • HTR 2A antibody
    • HTR2 antibody
    • HTR2, formerly antibody
    • HTR2A antibody
    • serotonin 5-HT-2 receptor, formerly antibody
    • serotonin 5-HT-2A receptor antibody
    • Serotonin receptor 2A antibody
    see all

画像

  • ab66049 at 1/100 dilution staining 5HT2A Receptor in Mouse frontal cortical neuron cultures (p1 pups, day 4 culture, for 48hrs) by Immunohistochemistry, Paraformaldehyde-fixed tissue.
  • Anti-5HT2A Receptor antibody (ab66049) at 1/400 dilution + Human Platelet (Human adult normal cell line) Whole Cell Lysate at 20 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 53 kDa
    Observed band size: 53 kDa
    Additional bands at: 57 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 4 minutes
  • ab66049, at 1/300 dilution staining 5HT2A Receptor in Rat cerebral cortex by Immunohistochemistry, Paraformaldehyde-fixed tissue.

参考文献

This product has been referenced in:
  • Szlachta M  et al. Repeated Clozapine Increases the Level of Serotonin 5-HT1AR Heterodimerization with 5-HT2Aor Dopamine D2Receptors in the Mouse Cortex. Front Mol Neurosci 11:40 (2018). Read more (PubMed: 29497362) »
  • Wang P  et al. Inflammatory mediators mediate airway smooth muscle contraction through a G protein-coupled receptor-transmembrane protein 16A-voltage-dependent Ca2+ channel axis and contribute to bronchial hyperresponsiveness in asthma. J Allergy Clin Immunol 141:1259-1268.e11 (2018). WB . Read more (PubMed: 28754608) »
See all 8 Publications for this product

レビューと Q&A

1-5 of 5 Abreviews or Q&A

Question

This customer has purchased ab66049 (Anti-5HT2A Receptor antibody) and has conducted the wb several times with rat sample. The results show no major band and high background, therefore, he would like to ask for your help to modify his experiment step, could you please offer any suggestion to improve his result?

I attached the image in this letter and her experiment step as follow:



1. Order details:


Batch number: gr55638-1

Po: 1028918

Abcam product code: ab66049

Antibody storage conditions (temperature/reconstitution etc) aliquot and stored at -20oC




2. Please describe the problem (high background, wrong band size, more bands, no band etc).

No major band and high background

3. On what material are you testing the antibody in WB?

· Species: SD rat, female

· What’s cell line or tissue: brain tissue; hippocampus & prefrontal cortex

· Cell extract or Nuclear extract:

· Purified protein or Recombinant protein: Purified



3. The lysate


How much protein was loaded: 10 microgram

What lysis buffer was used: self-made (sucrose, EDTA, and protease inhibitor cocktail)



What protease inhibitors were used: sigma (SI-P2714-1BTL)

What loading buffer was used: buffer was prepared with the following components of Tris-HCl (pH6.8), SDS, glycerol, beta-mercaptoethanol, EDTA, bromophenol blue

Phosphatase inhibitors

Did you heat the samples: temperature and time: 95oC for 5minutes, followed by 4oC-incubation for 3 mintues




4. Electrophoresis/Gel conditions/ Transfer conditions


Reducing or non reducing gel: reducing

Reducing agent: beta-mercaptoethanol

Gel percentage : 8% running gel

Transfer conditions: (Type of membrane, Protein transfer verified): PVDF, protein marker was completely transferred




5. Blocking conditions


Buffer: TBST

Blocking agent: milk, BSA, serum, what percentage: milk, 5%

Incubation time: 1 hour

Incubation temperature: room temperature




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:



7. Secondary Antibody


Species:Rabbit

Reacts against: mouse and rat

At what dilution(s) have you tested this antibody: 1/100 as suggested

Incubation time: over 14 hours at 4oC

Wash steps: membrane soaked in TBST and incubated in gentle shake for 5 minutes at room temperature; 3 times

Fluorochrome or enzyme conjugate: enzyme conjugate; HRP

Do you know whether the problems you are experiencing come from the secondary? Yes, and I see no problem with the secondary since it was applied on other antibody detection on the same membrane, and they all worked out, except anti-5HT 2A




8. Detection method
ECl, ECl+, other detection method: ECL



9. Did you apply positive and negative controls along with the samples? Please specify.



I only got the internal control (beta actin) for quantitative analysis

10. Optimization attempts

· How many times have you tried the Western? 3

· Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): Yes, since there was no any band found

· Do you obtain the same results every time e.g. are background bands always in the same place? The background was clear

· What steps have you altered? Primary antibody dilution



Could you please help this customer to solve the problem?

Thanks for your kindly help

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Answer

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimize the results from abab66049 Anti-5HT2A Receptor antibody. I would also appreciate if you can confirm some further details:


1. As this is a multipass membrane protein, which tent to aggregate in the loading buffer when boiled at high temperature due to their hydrophobic nature, I would like to recommend lowering the boiling temperature down to 60C and prolonging the boiling time up to 10 min. In addition, use DTT instead of beta-mercaptoethanol as this kind of aggregation is favoured by b- ME.

2. Actin is a reliable control for cytoplasmic proteins, but as you are searching for an membrane protein, I would suggest to use an appropriate membrane control.

3. In order to avoid under-loading, I would like to suggest loading 20 to 30 ug protein per lane.

4. Unfortunately, I could not find any information regarding the primaryantibody concentration and incubation set up. My guess is that this information can be found in the Secondary Antibody section. Could you please check this?

5.Have you stripped the membrane before you applied this antibody? If so, how often?

6. I am a bit confused regarding the secondary antibody: From how I understand it, you are using a rabbit secondary antibody against mouse andrat. Could you please confirm in which species the secondary antibody was produced and which species it detects?


In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I thank you for your cooperation.

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Answer

Thank you for contacting us.

Your credit note ID is 21936.

I am sorry that this antibody did not perform as stated on the datasheet. I have asked our accounting department to issue a credit note for you, which can be redeemed against the invoice of a future order by passing it on to your purchasing department. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used. If you have questions on how to use the credit note, please contact our accounting department.

If payment has already been made on the original order and you wish to receive a refund, please ask your purchasing department to contact our accounting department so that we may gather the correct information needed for the refund. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used.

Our accounting department can be contacted by email at us.credits@abcam.com or by telephone using the information at the Contact Us link in the top right corner of our website. Please refer to the credit note ID in any correspondence with our accounting department.

The credit note ID is for your reference only and does not automatically guarantee the credit.

I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service, should you require further expert advice.

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Question

What was the blocking buffer? 5% Non-fat milk, blocking for 2h. What were the antibodies incubated in? Plastic incubation box. What was the wash solution? TBS-T(0.1% Triton-100) What were the times and temperatures of each of these steps? 3 times , each time wash for 10 min. Were positive controls, loading controls used? The positive control is rat brain lysis which brought from Santa Cruz company. Finally, would you be able to provide me with the Abcam order id or PO used to purchase these products? I sent my order to the sectary in my department. Maybe I could give the number next week. Thank you for your help! Best,   Qing  2012/7/20 https://www.abcam.com/index.html?utm_campaign=CRM&utm_source=Abcam.CRM&utm_medium=Email&utm_term=Header Dear Dr Shu     Thank you for contacting us. I am very sorry to hear that you have been experiencing troubles when using our products. I would also like to thank you for providing me with details of you protocol. Our products are covered by Abpromise guarantee for a period of six months after delivery. Should this product not perform as indicated on the datasheet, we provide scientific support, replacement or refund. I was hoping that you could provide me with a few additional details about your experiments: What was the blocking buffer? What were the antibodies incubated in? What was the wash solution? What were the times and temperatures of each of these steps? Were positive controls, loading controls used? Finally, would you be able to provide me with the Abcam order id or PO used to purchase these products? More information on our Abpromise may be found at the following link: https://www.abcam.com/index.html?pageconfig=abpromise I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information. Use our products? Submit an Abreview. Earn rewards! https://www.abcam.com/abreviews Help us improve our service. https://www.abcam.com/index.html?pageConfig=technicalSurvey&intCCEID=3978058 Best regards, Keith Keith Beadle Scientific Support Specialist Abcam Inc. https://www.abcam.com Your original inquiry to Abcam: Name: Qing Shu Phone: tel:4023045746 Product code: 66049 Lot number: GR 28433-7   Inquiry: I can not detect the signal using the antibodies above. My sample is rat brain. I extracted the protein using RIPA buffer and the ratio is 1mg:5ul. After that put on ice for one hour and votexed them every 10min. Then centrifuge them with 16,000g for 15min and got the supernatant.Mix them with 2x loading buffer then boiled them for 6min at 100. I added 40ug protein totally into per lane running a 12% pre-cast gel. It took 30min at 80V running stacking gel and 60min at 120V running resolving gel. The dilute of ab21218 was 1:600 or 1:300 while the secondary dilute of antibody was 1:5000 or 1:10000. In addition , the ratio of ab66049 is 1:400 and the secondary dilute was 1:5000. I used a infrared imaging     Abcam Customer Services and Scientific Support Team https://www.abcam.com/index.html?pageconfig=technical&utm_campaign=CRM&utm_source=Abcam.CRM&utm_medium=Email&utm_term=Body [CCE3978058]     Discover more at http://abcam.com

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Answer

Thank you for your message and for providing this further information. I am sorry to hear these products did not work a stated on our datasheet. I appreciate the time you have spent on these experiments, and would like to offer a free of charge replacement (with either a new vial of these products or with new products), or if you would prefer, a refund/credit note in compensation (providing the product has been purchased in the last 180 days). In order to arrange this, I would appreciate if you could confirm your order number and date of purchase. I look forward to hearing from you when you are able to get this information. Thank you for your continued cooperation. I look forward to hearing from you with details of how you would like to proceed.

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Answer

Thank you for contacting us.

I am very sorry to hear that you have been experiencing troubles when using our products. I would also like to thank you for providing me with details of you protocol.Our products are covered by Abpromise guarantee for a period of six months after delivery. Should this product not perform as indicated on the datasheet, we provide scientific support, replacement or refund. I was hoping that you could provide me with a few additional details about your experiments:

What was the blocking buffer?

What were the antibodies incubated in?

What was the wash solution?

What were the times and temperatures of each of these steps?

Were positive controls, loading controls used?

Finally, would you be able to provide me with the Abcam order id or PO used to purchase these products?

More information on our Abpromise may be found at the following link:

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


I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

Read More

Question

To whom it may concern,
1. Order details:
- Batch number:
- Abcam product code: ab66049
- Antibody storage conditions (temperature/reconstitution etc) aliquot and stored at -20oC
2. Please describe the problem (high background, wrong band size, more bands, no band etc).
No band or smear
3. On what material are you testing the antibody in WB?
- Species: SD rat, female
- What’s cell line or tissue: brain tissue; hippocampus & prefrontal cortex
- Cell extract or Nuclear extract:
- Purified protein or Recombinant protein: Purified
3. The lysate
- How much protein was loaded: 10 microgram
- What lysis buffer was used: self-made (sucrose, EDTA, and protease inhibitor cocktail)
- What protease inhibitors were used: sigma (SI-P2714-1BTL)
- What loading buffer was used: buffer was prepared with the following components of Tris-HCl (pH6.8), SDS, glycerol, beta-mercaptoethanol, EDTA, bromophenol blue
- Phosphatase inhibitors
- Did you heat the samples: temperature and time: 95oC for 5minutes, followed by 4oC-incubation for 3 mintues
4. Electrophoresis/Gel conditions/ Transfer conditions
- Reducing or non reducing gel: reducing
- Reducing agent: beta-mercaptoethanol
- Gel percentage : 8% running gel
- Transfer conditions: (Type of membrane, Protein transfer verified): PVDF, protein marker was completely transferred

5. Blocking conditions
- Buffer: TBST
- Blocking agent: milk, BSA, serum, what percentage: milk, 5%
- Incubation time: 1 hour
- Incubation temperature: room temperature
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:
7. Secondary Antibody
- Species:Rabbit
- Reacts against: mouse and rat
- At what dilution(s) have you tested this antibody: 1/100 as suggested
- Incubation time: over 14 hours at 4oC
- Wash steps: membrane soaked in TBST and incubated in gentle shake for 5 minutes at room temperature; 3 times
- Fluorochrome or enzyme conjugate: enzyme conjugate; HRP
- Do you know whether the problems you are experiencing come from the secondary? Yes, and I see no problem with the secondary since it was applied on other antibody detection on the same membrane, and they all worked out, except anti-5HT2A
8. Detection method
ECl, ECl+, other detection method: ECL
9. Did you apply positive and negative controls along with the samples? Please specify.
I only got the internal control (beta actin) for quantitative analysis
10. Optimization attempts
- How many times have you tried the Western? 3
- Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): Yes, since there was no band found
- Do you obtain the same results every time e.g. are background bands always in the same place? The background always remained clear with no any band
- What steps have you altered?

PS the area framed by red box on the attached figure represents the 3 independent membranes for 5HT2A detection

Appreciate your assistance

Read More
Answer

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab66049 :

I would load slightly more sample on the gel, up to 30µg. This may help.
When testing our antibodies, our lab uses 5% BSA as a blocking reagent, so I recommend switching to this instead of milk. Some antibodies give stronger, more specific signals on blots blocked with BSA instead of milk, so doing this may improve the signal you are seeing. An example of the above is the western blot obtained with the Abcam GAPDH antibody ab9385 : https://www.abcam.com/ab9385.



Should the suggestions not improve the results, please do let me know.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within4 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

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

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