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Dear tech, A customer has some questions about ab109878 and ab109911 .As follows: 1. Whether samples extracted from tissue with solution 1 provided in the kit can used to perform WB experiment for other proteins expressing in membrane, cytoplasmic,and nucleus? 2.Whether solution 1 contains protease inhibitors or phosphatase inhibitor? 3.As the tissues are small, whether the cutomer could use 200ul solution 1 to extract 1mg tissue? I would like to apologize for having taken so much of your time.Thank you for your help. Best regards,
Asked on Oct 19 2011
Thank you for contacting us. 1. The detergent used in this kit is not efficient at extracting nuclear samples. Most of the nuclear proteins will be in the pellet obtained after the centrifugation step during the sample preparation (as explained in the protocol). Other membrane and cytoplasmic proteins can be found in the extract obtained with the kit ab109878, however these proteins will be in their native form. It will be necessary to mix this extract with 2XSDS buffer before running the samples on a western blot. 2. Solution 1 does not contain protease inhibitors. In the additional materials required section of the protocol we suggest the use of protease inhibitors. 3. My suggestion is to use between 1 and 5mg of wet tissue in 100 - 200uL of buffer during the homogenization. Protein concentration in the homogenate should be greater than 1mg/mL. With such a small sample I suggest to use a very small glass dounce homogenizer (0.5mL). I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
Answered on Oct 19 2011