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The gels that we have been using are non-reducing, non-denaturing. The amount of protein loaded vary according to our daily plans.... but should be more than sufficient in order to detect bands. We haven't used loading controls at this point... we wanted to see something first. We used the NP-40 buffer documented in your western blot beginners pdf file. In the case of all the antibodies ordered, they have been designated by Abcam as tested for Western blots. Do you have any images on files that I can take a look at? What type of tissue did you use for the testing? How different was your protocol from the one established in our lab? Sorry for all the questions but maybe I can use this info for some of the trouble shooting on our end.
Asked on Jul 25 2006
Thank you for your enquiry. I appreciate your continued patience in this matter. I have received some feedback from some of the sources of the antibodies that you are enquiring about; ab7033 - To follow on with the comments that I have made the source of mouse monoclonal [MMP2/2C1] to MMP2 (ab7033) was also concerned with regards the approach that you have used for your sample preparation. I appreciate that you have been using an NP40 buffer extraction. However, this is an approach designed for a cytoplasmic preparation of cell culture cells. I would like to follow up my previous email by recommending that you perform a loading control experiment using an antibody that targets a "housekeeping protein" for example GAPDH or beta actin. This can be performed under the conditions best recognized by the antibody you are using; most likely denaturing, reduced. This will enable you to fully determine the integrity of your protein with respect to its protein composition. My biggest concern with the blot images that you have provided me with are the band doublet that you have been detecting either side of the 37KDa marker as this is present in the majority of your blot images. The source of ab6586 - Collagen IV antibody (ab6586) makes a similar suggestion although Collagens should in fact be electrophoresed as you have been using non-denaturing, non-reduced conditions. However, it is important that the integrity of your samples are confirmed. I am still awaiting further information and am in the process of requesting blot images for the antibodies that you have enquired about. I appreciate your continued patience. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.
回复于 Aug 01 2006