Overview

  • Product name

  • Description

    Rabbit polyclonal to MC2-R
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WBmore details
  • Species reactivity

    Reacts with: Human
    Predicted to work with: Mouse, Rat, Rabbit, Horse, Guinea pig, Cat, Dog
  • Immunogen

    Synthetic peptide corresponding to Human MC2-R aa 86-135 (N terminal). (NP_000520.1)
    Sequence:

    LRNMGYLKPRGSFETTADDIIDSLFVLSLLGSIFSLSVIAADRYITIFHA

  • Positive control

    • Human fetal kidney lysate.
  • General notes

    Protein previously labeled as MC2 receptor.

Properties

Applications

Our Abpromise guarantee covers the use of ab125398 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 Use a concentration of 1 µg/ml. Predicted molecular weight: 34 kDa. Good results were obtained when blocked with 5% non-fat dry milk in 0.05% PBS-T.

Target

  • Function

    Receptor for ACTH. This receptor is mediated by G proteins (G(s)) which activate adenylate cyclase.
  • Tissue specificity

    Melanocytes and corticoadrenal tissue.
  • Involvement in disease

    Defects in MC2R are the cause of glucocorticoid deficiency type 1 (GCCD1) [MIM:202200]; also known as familial glucocorticoid deficiency type 1 (FGD1). GCCD1 is an autosomal recessive disorder due to congenital insensitivity or resistance to adrenocorticotropin (ACTH). It is characterized by progressive primary adrenal insufficiency, without mineralocorticoid deficiency.
  • Sequence similarities

    Belongs to the G-protein coupled receptor 1 family.
  • Cellular localization

    Cell membrane.
  • Information by UniProt
  • Database links

  • Alternative names

    • ACTH receptor antibody
    • ACTH-R antibody
    • ACTHR antibody
    • ACTHR_HUMAN antibody
    • Adrenocorticotropic hormone receptor antibody
    • Adrenocorticotropin receptor antibody
    • Corticotropin receptor antibody
    • MC2 receptor antibody
    • MC2-R antibody
    • MC2R antibody
    • Melanocortin 2 receptor (adrenocorticotropic hormone) antibody
    • Melanocortin 2 receptor antibody
    • Melanocortin receptor 2 antibody
    see all

Images

  • Anti-MC2-R antibody (ab125398) at 1 µg/ml + Human fetal kidney lysate at 10 µg

    Predicted band size: 34 kDa



    Gel concentration: 12%.

References

ab125398 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Question

If you could send me the other mc2r antibody, ab77347, it would be great. I hope I have better luck with that one.























https://www.abcam.com/index.html?utm_campaign=CRM&utm_source=Abcam.CRM&utm_medium=Email&utm_term=Header









Dear Annika

I agree that if you are not seeing anything between 30 and 40 kDa then the antibody is probably the issue, and not the sample preparation. If you want to try ab77347 as a free-of-charge replacement for ab125398 (you do not need to send back the remainder of ab125398), please let me know. I think the western blot data we have for ab77347 is more convincing thanour blot for ab125398, since the MC2R precursor is 34 kDa, and the post-translation glycosylations will cause the protein to run more slowly in the gel. That may be whythe bandappears at 40 kDa in the ab77347 blot.

The adrenal lysate probably has less GAPDH as a percentage of total protein than the cell lysates do, since ab29249 is a tissue lysate, containing matrix proteins in addition to cellular proteins. That would be one explanation forwhy there is apparently less GAPDH in that lane, if you loaded equal amounts of total protein. In addition, different cells will have different amounts of protein.

In general, loading control antibodies like anti-GAPDH are useful when comparing samples of a single cell or tissue. Loading control antibodiesarenot a valid wayofconfirming even loading of samples from different cells or tissues.




Best regards,
Thomas

Thomas Ruyle
Scientific Support Specialist
Abcam Inc.
https://www.abcam.com


Your original inquiry to Abcam:





Thanks for the suggestion, I will try heating the samples no more than 70 degrees. Although I am afraid this is not the problem since I don’t detect any band at all between 30 and 40 kDa.



Yes, the weaker band in the adrenal lysate was for the GAPDH blot. This is why I was thinking the adrenal lysate might have been degraded. I can’t say the band looked more blurry, just more faint and weaker compared to the other bands. Please see the attached image of the GAPDH western, the adrenal lysate is the sample to the right.



I have stained the gel to check the efficacy of the transfer and it was ok. Moreover, the GAPDH blot was done on the same membrane as the MC2R.



Do you have any suggestions of other positive controls I could use? I know abcam has another antibody for MC2R, could it be better?



Best regards,

Annika



……………………………………………………………….

Annika Lindskog Jonsson, PhD student

The Renal Center

The Wallenberg laboratory for Cardiovascular Research

Sahlgrenska University Hospital

Bruna Stråket 16,

413 45 Göteborg, Sweden

Phone: +46 31342 8490

Fax: +46 3182 37 62







Från: Kerstin Ebefors
Skickat: den 3 april 2012 21:24
Till: Annika Lindskog Jonsson
Ämne: VB: Reply from Abcam to your enquiry regarding ab125398 [CCE3655800]











Från: mailto:technical@abcam.com mailto:[mailto:technical@abcam.com]
Skickat: den 3 april 2012 21:23
Till: Kerstin Ebefors
Ämne: Reply from Abcam to your enquiry regarding ab125398 [CCE3655800]



















https://www.abcam.com/index.html?utm_campaign=CRM&utm_source=Abcam.CRM&utm_medium=Email&utm_term=Header









Dear Kerstin

Thank you for completing our troubleshooting form. Your protocol looks correct. My one concern is that the receptor may be aggregating when you heat the samples to 95C. MC2R, like other multi-pass membrane receptors, tends to formaggregates when boiled, and to avoid this,many researchers heat samples that contain multi-pass membrane proteins no higher than 70C. The aggregates either do not enter the gel, or do not transfer to the membrane.

Still, I think you should see a band between 30 and 40 kDa,especially for the adrenal lysate.I do not know what the protein at 70 kDa for the kidney sample could be.

You mentioned in your report that "...the adrenal lysate showed a weaker band compared to the other ones". Are you referring to the GAPDH blot? If so, does the band look more blurry that the GAPDH band of the other samples? It is possible it is degraded.

Did you do a stain of the membrane before the antibody, for instance a Ponceau Red stain, to check for the efficacy of the transfer?




Help us improve our service.
https://www.abcam.com/index.html?pageConfig=technicalSurvey&intCCEID=3655800


Best regards,
Thomas

Thomas Ruyle
Scientific Support Specialist
Abcam Inc.
https://www.abcam.com


Your original inquiry to Abcam:


LOT NUMBER GR77375-1 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE Protein from kidney tissue, podocytes, endothelial cells and whole cell adrenal lysate (ab29249) as positive control. PRIMARY ANTIBODY Dilution 1:1000 in blocking buffer. 1 hour room temperature. DETECTION METHOD ECL kit used for detection. Exposure for up to 1 hour. POSITIVE AND NEGATIVE CONTROLS USED Positive control whole cell adrenal lysate (ab29249) ANTIBODY STORAGE CONDITIONS aliquoted and stored in -20 SAMPLE PREPARATION Protease inhibitors in lysis buffer. Samples heated in 95 degrees 5 minutes. AMOUNT OF PROTEIN LOADED 12 µg ELECTROPHORESIS/GEL CONDITIONS Reducing conditions, 4-12 % bis-tris gel. TRANSFER AND BLOCKING CONDITIONS TBS-Tween + 5% non fat milk used for blocking and dilution. Blocking over night +4. SECONDARY ANTIBODY Wash in TBS-T 3x5 minutes before secondary antibody (anti-rabbit) diluted 1:5000. After incubation 1 hour room temperature, wash in TBS-T 3x5 minutes. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes ADDITIONAL NOTES The only band that is detected is one at around 70kDa in the kidney sample. But the MC2R band should be present around 30-40 kDa. No band is visible in the adrenal sample which should be the positive control. Do you have any suggestions of other positive controls I could use? I have run a GAPDH antibody with all these sampless, since it should be stably expressed. Although equal amount of protein was added to the wells, the adrenal lysate showed a weaker band compared to the other ones. Could the adrenal lysate have been degraded? Immediately upon arrival it was aliquoted and stored in -80.




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

[CCE3655800]











Discover more at abcam.com





























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

[CCE3658745]











Discover more at abcam.com

Read More
Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacemen, the other MC2R antibody, ab77374.

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.

Read More

Answer

I agree that if you are not seeing anything between 30 and 40 kDa then the antibody is probably the issue, and not the sample preparation. If you want to try ab77347 as a free-of-charge replacement for ab125398 (you do not need to send back the remainder of ab125398), please let me know. I think the western blot data we have for ab77347 is more convincing than our blot for ab125398, since the MC2R precursor is 34 kDa, and the post-translation glycosylations will cause the protein to run more slowly in the gel. That may be why the band appears at 40 kDa in the ab77347 blot.

The adrenal lysate probably has less GAPDH as a percentage of total protein than the cell lysates do, since ab29249 is a tissue lysate, containing matrix proteins in addition to cellular proteins. That would be one explanation for why there is apparently less GAPDH in that lane, if you loaded equal amounts of total protein. In addition, different cell lines will have different amounts of GAPDH protein.

In general, loading control antibodies like anti-GAPDH are useful when comparing samples of a single cell line or tissue. Loading control antibodies are not a valid way of confirming even loading of samples from different cell lines or tissues.

Read More

Question

DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE Protein from kidney tissue, podocytes, endothelial cells and whole cell adrenal lysate (ab29249) as positive control. PRIMARY ANTIBODY Dilution 1:1000 in blocking buffer. 1 hour room temperature. DETECTION METHOD ECL kit used for detection. Exposure for up to 1 hour. POSITIVE AND NEGATIVE CONTROLS USED Positive control whole cell adrenal lysate (ab29249) ANTIBODY STORAGE CONDITIONS aliquoted and stored in -20 SAMPLE PREPARATION Protease inhibitors in lysis buffer. Samples heated in 95 degrees 5 minutes. AMOUNT OF PROTEIN LOADED 12 µg ELECTROPHORESIS/GEL CONDITIONS Reducing conditions, 4-12 % bis-tris gel. TRANSFER AND BLOCKING CONDITIONS TBS-Tween + 5% non fat milk used for blocking and dilution. Blocking over night +4. SECONDARY ANTIBODY Wash in TBS-T 3x5 minutes before secondary antibody (anti-rabbit) diluted 1:5000. After incubation 1 hour room temperature, wash in TBS-T 3x5 minutes. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes ADDITIONAL NOTES The only band that is detected is one at around 70kDa in the kidney sample. But the MC2R band should be present around 30-40 kDa. No band is visible in the adrenal sample which should be the positive control. Do you have any suggestions of other positive controls I could use? I have run a GAPDH antibody with all these sampless, since it should be stably expressed. Although equal amount of protein was added to the wells, the adrenal lysate showed a weaker band compared to the other ones. Could the adrenal lysate have been degraded? Immediately upon arrival it was aliquoted and stored in -80.

Read More
Answer

Thank you for completing our troubleshooting form. Your protocol looks correct. My one concern is that the receptor may be aggregating when you heat the samples to 95C. MC2R, like other multi-pass membrane receptors, tends to form aggregates when boiled, and to avoid this, many researchers heat samples that contain multi-pass membrane proteins no higher than 70C. The aggregates either do not enter the gel, or do not transfer to the membrane.

Still, I think you should see a band between 30 and 40 kDa, especially for the adrenal lysate. I do not know what the protein at 70 kDa for the kidney sample could be.

You mentioned in your report that "... the adrenal lysate showed a weaker band compared to the other ones". Are you referring to the GAPDH blot? If so, does the band look more blurry that the GAPDH band of the other samples? It is possible the sample is degraded.

Did you do a stain of the membrane before the antibody, for instance a Ponceau Red stain, to check for the efficacy of the transfer?

Read More

Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
For licensing inquiries, please contact partnerships@abcam.com

Sign up