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Joe101

Member Since 12 Apr 2012
Offline Last Active May 12 2013 04:37 AM
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Topics I've Started

Primer Efficiency across runs

08 May 2013 - 10:24 AM

Hi,

When running serial dilutions for testing primer efficiency, in this case for use with the Pfaffl method, do they need to be run on the same plate as the samples you are testing?  I had allways thought of primer efficiency beign a property of the primer design, but I guess this must interact with running conditions to a certain extent.

Thanks,
Joe

Determining protein concentration after solubilisation in Triton X-100

21 February 2013 - 03:08 AM

Hi,

The protocol I'm using solubilises brain tissue in 5% Triton X-100 before determining protein concentration buying the Bradford protein assay (for use in SDS-PAGE). I have read however that detergents such Triton X-100 can interfere with the assay;  I only need an estimate for gel loading, does anyone know if the presence of this high a level of Tritxon X-100 will significantly effect the results, and if so, suggest any alternatives to the Bradford assay?

Many Thanks,
Joe

General chat about tween-20 as a WB block and wash

18 January 2013 - 04:01 AM

Hi,

I was interested about the specific mechanism by which tween-20 works as a block and wash of western blot membranes, but unfortunately I couldn't find much on it except for one bulletin. This suggested  the large size of tween-20 would make it useful for a block but also that it's size is due to it's large hydrophillic head so that doesn't quite make sense. As for it's ability to wash the membrane of antibodies, tween-20 has a low CMC and so may incorporate loosely bound antibodies into mixed micelles, while leaving bigger more strongly bound proteins on the membrane.

I dont know though... any ideas?

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