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Please provide a western blot image for ab3559 and ab3558
Asked on Apr 22 2008
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.
Answered on Apr 22 2008