Anti-Cannabinoid Receptor I抗体(ab3558)

Thank you for contacting us.

We have following CB1 and CB2 available. These are testes in flow cytometry and are fully guaranteed.

ab3558; https://www.abcam.com/Cannabinoid-Receptor-I-antibody-ab3558.html

ab3561; https://www.abcam.com/Cannabinoid-Receptor-II-antibody-ab3561.html

ab3560; https://www.abcam.com/Cannabinoid-Receptor-II-antibody-ab3560.html

Could you let me know which conjugated you would like for compatible secondary antibody?

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

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Thank you for your inquiry.

The best way to find isotype controls for an antibody is to start at our homepage www.abcam.com. Go to the yellow bar at the top of the page and move the mouse to “Products,” then click on “Isotype Controls”. The direct link is:

https://www.abcam.com/index.html?pageconfig=catalog_byproducttype&intProductTypeID=20

Then select the correct isotype control based on the host species as well as the isotype, sub-isotype, conjugation, and application of the primary antibody which you are using.

For example, if the primary antibody is a FITC-conjugated mouse IgG1 for use in flow cytometry, then you will need to choose a FITC-conjugated mouse IgG1 isotype control tested in flow cytometry.

Please note that most isotype controls are monoclonal. They are not suitable for use with polyclonal antibodies as these contain more than one IgG subclass. However, we do provide several polyclonal isotype controls for use in these circumstances.

I taken the information that you have provided me to determine that the best isotype control in this instance is ab37415 Rabbit IgG- Chip Grade. Should you decide to conjugate a fluorescent tag onto your antibodies however,it is advised that you use a different isotype control which is also conjugated to the same fluorescent label.

Please let me know if have any questions. I would be happy to assist you.

Thank you for your enqiury. According to our most recent internal testing data, by Western blot, ab3558 Cannabinoid Receptor I antibody shows detection of an ~ 52 kDa band in rat brain homogenate. After further research on anti-Cannabinoid Receptor 1 ab3558 , some recently published articles have shed light on the molecular weight. Reference 1 shows this antibody detecting CB1 at ~50 kDa (see reference 1 below). However, a more recent reference shows our antibody detecting CB1 expressed in macaque cortical tissue homogenate at a molecular weight of ~85 kDa and ~52 kDa (see reference 2 below). Therefore, according to the literature, it appears that ab3558 Cannabinoid Receptor I antibody will recognize CB1 at two different molecular weights, ~50 kDa and ~85 kDa. References: Reference 1: Citation: J. Neurosci., December 3, 2003. 23(35):11136-11141 Link: http://www.jneurosci.org/cgi/reprint/23/35/11136 Description: This article cites ABR's anti-Cannabinoid Receptor 1 (PA1-743) being used in WB on temporal cortex from human AD patients. "Western blotting. The protocol used is basically as described previously (Romero et al., 2002). Human brain was obtained at autopsy and a 1 gm piece of cerebral cortical gray matter was homogenized in 10 ml of M-PER mammalian protein extraction reagent. The homogenate was shaken gently for 10 min and then centrifuged at 27,000 x g for 15 min. The supernatant was isolated, and protein was determined using the BCA protein assay kit. Brain protein extract (50 µg) was reduced and denatured and separated by electrophoresis through a 10.5 x 10 cm, 0.75-mm-thick 15% polyacrylamide preparative gel. After separation, the proteins in the gel were transferred to nitrocellulose membrane. The nitrocellulose was washed with PBS containing 0.2% Tween 20 (PBST), and remaining binding sites on the membrane were blocked by overnight incubation in PBST containing 2% nonfat dried milk at 4°C. Incubation of primary antibodies was performed at 1:300 dilution in PBST containing 2% nonfat dried milk overnight at 4°C. In some experiments, the antibodies were preincubated with 8 µg/ml of the same immunizing peptides used for the generation of the antibodies. After the nitrocellulose membrane was washed with PBST, it was incubated with an alkaline phosphatase-conjugated goat anti-rabbit secondary antibody (Sigma, St. Louis, MO), 1:2000 in PBST containing 2% nonfat dried milk for 1 hr at room temperature. The nitrocellulose membrane was washed extensively with PBST, followed by PBS. Finally, the immune complex was visualized by incubating in the presence of nitroblue tetrazolium-5-bromo-4-chloro-3-indoyl phosphate chromogen." The images can be found in Figure 1 on page 11138. Reference 2: Citation: J. Neuro. 25(10):2530-2536, 2005. Link: http://www.jneurosci.org/cgi/reprint/25/10/2530 Description: This article cites ABR's PA1-743 antibody detecting CB1 at ~85 kDa and ~52 kDa in macaque cortical tissue homogenate. "Western blotting. Protein extracts were prepared from frontal cortices of one uninfected control and two SIVE monkeys that were used for immunohistochemistry. Tissues were homogenized in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40) with 1x protease inhibitor mixture. The homogenate was incubated on ice for 30 min and then centrifuged at 10,000 x g for 30 min at 4°C. The supernatant was collected, and protein concentration was determined using the BCA protein assay kit. Twenty-five micrograms of protein extract from each sample were reduced and denatured and separated by electrophoresis through a 4-15% gradient polyacrylamide preparative gel. The proteins were transferred from the gel to Immuno-Blot polyvinylidene difluoride membrane. The membrane was washed with TBS containing 0.1% Tween 20 (TBST) and blocked in TBST containing 5% dry goat milk. Primary antibody (ab3558) was diluted in TBST containing 5% dry goat milk at 1:2000 for CB1; this was incubated overnight at 4°C with gentle shaking. Blots were washed four times in TBST and then incubated with goat anti-rabbit HRP (1:1000) for 1 h at room temperature. The membrane was washed four times in TBST. Finally, the immune complex was visualized using an ECL Western Blotting kit. The specificity of the signal was confirmed by preincubation with the immunizing peptide (1:900)." The images can be found in Figures 1 & 2 on page 2532. With regards to ab3559 Cannabinoid Receptor I antibody, although we do not have an image available from our testing data, I have provided the original reference for this antibody. I have listed this paper as well as another reference below for your review. The paper has a wonderful western blot image on page 395 (Fig. 1 A and B) showing detection of CB1 in Sprague-Dawley rat brain extracts. References: K. Tsou et. al. Immunohistochemical Distribution of Cannabinoid CB1 Receptors in the Rat Central Nervous System. Neuroscience. 1998 Mar;83(2):393-411 Andrea L. Small-Howard et. al. Anti-inflammatory potential of CB1-mediated cAMP elevation in mast cells. Biochem. Journal 25 Jan. 2005 manuscript BJ0041682. http://www.biochemj.org/bj/imps_x/pdf/BJ20041682. I hope that this information is helpful. Please do not hesitate to contact me with any additional questions.

Thank you for your message which has been forwarded to me on behalf of Tanya while she is away. It is with regret that we are not able to guarantee this product if it has not been stored as indicated on the datasheet. However, the results indicate that the antibody is still active and it is very likely the bands observed are from splice variants of the cannabinoid receptor 1 protein. There are splice variants of the protein in rat as well as human as indicated in the following reference: Journal of Pharmacology and Experimental Therapeutics. Vol. 282, Issue 3, 1632-1642, 1997 Regional Differences in Cannabinoid Receptor/G-protein Coupling in Rat Brain1. Christopher S. Breivogel et al. The extra band could also result from differences in post-translational modification. I hope this information is helpful. Should the customer have any further questions, please do not hesitate to contact us again.

Thank you for your email. I'm sorry that this antibody is not working out under your conditions and I have instructed our accounting department to issue a refund for your order for ab3558. If you have any questions regarding the refund, please contact accounts@abcam.com

Thank you for your email and patience. I do suggest decreasing the concentration of the primary as well as the incubation period as the bands you are seeing may be non-specific. If you are using the same secondary with the Pten primary and not getting any additional bands then your secondary is most likely working fine. However, you probably still need to optimize the conditions with ab3558.

Thank you for your email. Yes, this has been tested and characterized in Western blotting as well as Immunocytochemistry. In Western blotting, this antibody detects an ~60 kDa protein representing CB1 from rat brain homogenate. The bands that you are seeing at 45 and 80 kDa may be non specific and this point I would suggest decreasing the concentration of the primary as well as the incubation period. Also, make sure to run a secondary control to ensure that these bands are not due to your secondary antibody.

Thank you for your email. I recall that you were previously unable to obtain any bands with this antibody. I would like to investigate this present issue further but do need details regarding your protocol in order to do that. If you could answer the questions below, it would be very helpful. 1. Please describe the problem (high background, wrong band size, more bands, no band etc). 2. On what material are you testing the antibody in WB? •Species? •Cell extract/ Nuclear extract? •Purified protein? •Recombinant protein? 3. How much protein did you load? •How did you prepare the lysate for the analysis (protease inhibitors etc)? •Did you heat the samples? 4. Primary Antibody •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation time, wash step? 5. Secondary Antibody •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation time, wash step? •Do you know whether the problems you are experiencing come from the secondary? 6. What detection method are you using? 7. Background bands •Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control) •Is the blocking step sufficient? (We recommend blocking the membrane by adding 20 ml of blocking buffer (5% non-fat dry milk, 0.1% Tween-20 in TBS). Incubate for 2 h at room temperature or overnight at 4°C with agitation) •Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) •At what size are the bands migrating? Could they be degradation products of your target? •Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) 8. Optimization attempts •How many times have you tried the Western? •Do you obtain the same results every time e.g. are background bands always in the same place? •What steps have you altered? 9. Did you apply positive and negative controls along with the samples? Please specify.

There have been no previously reported problems with this particulat lot. It is a possibility that the antibody became defective during shipment. We will send you a replacement vial free of charge, please let me know how it works out for you.