Anti-UCP3 antibody (ab10985)

Thank you for sending on these details. The UCP3 protein that this antibody recognises undergoes a lot of post transcriptional modification events. The bands seen in the western blot are probably from the heavier glycosylated forms of the protein, which appear to be slightly different in muscle tissue and liver. Positive controls run using this antibody have shown bands at approximately 50kD. If the customer would like to be sure of this, they can de-glycosylate the protein before denaturing and running on the gel. (there are kits available for this if they do a search.) The bands should then run at approximately 34kD. I hope this helps. Please get back to me if there are any more questions.

Thank you for contacting us and taking the time to fill out the questionaire. I am sorry you have had trouble using this rabbit polyclonal anti-UCP3 antibody (ab10985) I have had a look at your procedure and have come up with the following suggestions to optimise your protocol which I hope will solve the problem. 1. Sample buffer Multiple band and bands of incorrect size can occur when the protein polymerises eg to dimers (which will run down the gel at a different speed). This can be prevented by ensuring adequate denaturing. Check that your loading buffer contains SDS for denaturing the protein and you could try heating for 10 minutes rather than 5 minutes. The samples should then be placed immediately on ice and run on the gel as soon as possible before the protein starts to form multimers again. 2. You have not mentioned how long you block for. It may be worth optimising your blocking step. You can try blocking overnight, or blocking at 4oC for shorter period of time. 3. The non specific binding could be due to excess of antibody. If this is the case,the suggested dilution on the datasheet is 1:1000, it would be better to titrate the antibody more dilute than this rather than less dilute as you have been doing. Your secondary antibody may also need titrating to prevent non specific binding. 4. I would not recommend you load any more than 100ug of protein per lane as this is fairly high. This will increase risk of multimers forming. 5. For your positive control, you could use a stronger lysing agent such as RIPA buffer (50mm TRis HCl, ph8, 150mM NaCl, 1% NP-40, 0.5%sodium deoxycholate, 0.1%SDS) and check the protien concentration in your tissue sample preparation to make sure there is enough protein. I hope this helps. Good luck with your experiment, and please let me know how you get on. If you have any more questions, or need any more advice, please contact me again. With thanks.