I am working with lungs from mice, and I want to do a western blot analysis to my samples. I am having trouble with this because my images turn out very "messy", with a high background. Perhaps I should filter my samples following their homogenization? Has anyone had this problem and knows how to avoid it? What is the best method to homogenize the samples?


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Doing westerns with primary tissue can be tough, especially because of the presence (sometimes at high levels) of extracellular matrix (ECM). This material can be quite resistant to homogenization and some lysis buffers. One method I have found to work is to snap-freeze the tissue in liquid nitrogen (not on dry ice or at -80°C) directly after removal from the animal, or after any sub-dissection you may wish to do. Then, when you are ready to prepare your sample, grind it thoroughly with a mortar and pestle in LN2, collect the dust and add a good lysis buffer like RIPA at 2-3X concentration, along with protease and phosphatase inhibitors, also at 2-3X. Then, instead of simply homogenizing, use a probe-tip sonicator (not a water bath one) and sonicate the lysate on ice with 8-10 pulses of perhaps 3-5 seconds each, ensuring that the lysate does not heat up too much, and making sure it does not froth up by getting the tip too close to the surface. Sonication sends very powerful shearing forces through liquid and is excellent at destroying membranes and breaking apart ECM. From here you can do your protein concentration assay (remember to use the same concentration of lysis buffer as your blank), aliquot the lysate and store at -80, or add SDS sample buffer and run your gel.

I routinely use sonication on all of my western samples, regardless of whether it's tissue or cell lines I'm dealing with, and I almost never get smeared lanes anymore. Sonication reduces the viscosity or "thickness" of the lysate, allowing you to pipette it much more easily and accurately. It is also useful when your sample "coagulates" after adding SDS sample buffer, and in this instance it doesn't matter if the sample gets hot, as you'll just be boiling it anyway.

I hope this helps, please let me know if you're still having problems. I used to do troubleshooting like this for a living :)


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