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Protein from mouse and human brain was extracted using a lysis buffer containing 150mM NaCl, 50mM Tris pH 7.5 and 0.1% Triton. Sonicated briefly and centrifuged to remove cell debris. 45ug of total protein was denatured in NuPage LDS sample buffer with 2-mercaptoethanol and then heated to 85 degrees before loading onto a NuPage 4-12% Bis- Tris Gel. The gel was run for 2hrs at 130mA. Protein was transferred to Immobilon-P membrane and blocked with 5% milk TBST for one hour. The membrane was then probed with the LRRTM3 ab in 5%milk TBST overnight at a concentration of 1ug/ml The next morning the membrane was washed 3X 10 min wash in TBST buffer then probed for 1 hr with an Invitrogen Rabbit HRP conjugated secondary Ab in 5% milk TBST. Membranes were then washed 3X 10mins in TBST. I have attached an image of the Western Blot and can provide a lighter exposure if required, but as you will see there is no difference between the WT and KO mouse brain tissue. This mouse KO mouse model has been published and we know from mRNA work that there should be no LRRTM3 expression. Lanes 1 Wild type mouse brain lysate 2 Heterozgous LRRTM3 knockout brain lysate 3 Homozygous LRRTM3 knockout brain lysate 4 Wild type mouse brain lysate 5 Heterozgous LRRTM3 knockout brain lysate 6 Homozygous LRRTM3 knockout brain lysate 7 Human brain cerebellum 8 Human brain temporal cortex 9 HEK293T cell line overexpressing LRRTM3 I will certainly take a look at the Abreview page. I look forward to hearing from you and please let me know if you need any further details.
Asked on Feb 14 2012
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Answered on Feb 14 2012