Hi!
A few months ago, I was troubleshooting human brain white matter protein extraction for western blot use. It turned out our chemicals were bad- upon replacement, everything worked. Now, we are beginning a massive white matter western blot study. Upon protein extraction of six subjects' cingulum bundle, the protein concentration was incredibly low (1 ug/ml) where we expected approximately 60ug/ml based on our previous refinement of the protocol. Initially, we believed there was an issue with our lysis buffer and made fresh solution. However, with the fresh solution, we still do not achieve the expected protein concentration, suggesting there's a problem at some point in the protocol or with one of our chemicals. If anyone could help, I would really appreciate it! Below is our protocol.
We use a 500ul/0.1g tissue concentration for lysis buffer. Our lysis buffer contains Trizma base, hydrochloric acid, EDTA, and sodium chloride. We store it at 4 degrees Celsius (for no longer than 2 weeks- after that we trash it and make fresh) and pH it to 8.0. Immediately before use we add SDS (1% concentration) and protease inhibitor (10ul/ml). We add the specific amount of lysis buffer based on the tissue weight to the tissue, and then sonicate it. After sonication, we allow the tissue to agitate at room temperature for 15 minutes to allow optimal protein extraction. Then, we centrifuge the homagenate at 4 degrees Celsius at 13,500 RPMs for 15 minutes. After this, we draw off the supernatant (leaving the pellet undisturbed). We then create a blank and two vials per sample to be used for spectrophotometry analysis. The blank contains 1097.5ul ultra pure water and 2.5ul of supernatant (usually whichever subject we have the most supernatant from). The subject vials contain 2.5ul supernatant, 197.5ul ultra pure water, 100ul of reagent A and 800ul reagent B (both from bio rad for DC protein assay). We allow these vials to react in the dark at room temperature for 60 minutes. We then used a modified Lowry method to analyze protein content via spectrophotometer.
I suspect the lysis buffer is made wrong (iff it worked previously!). There are many more traditional ways to obtain protein from brain for use in Westerns, the simplest is just using SDS-PAGE sample buffer to extract and running it directly, although this would make it impossible to check protein concentration and normalize sample loading. Many use RIPA buffer, see here for a relevant discussion:
https://www.researchgate.net/post/What_lysis_buffer_is_best_for_extracting_proteins_from_fresh_mouse_brain_tissue_for_western_blot1
Edward, I also believed that to be the case. However, after the initial failure, I made fresh lysis buffer using a recipe of 1.59g HCl, 0.74g Trizma base, 0.46g EDTA, and 2.19g of NaCl. I then refrigerate it to 4 degrees Celsius, and pH it to 8.0 using the HCl and Trizma base. Is there an isolated way to test the validity of the buffer?
The buffer sounds fine, you should begin thinking about it in terms of molarity as without the final volume (100mL, 1L ?) I have no way of knowing the final concentrations. Realize the HCl is only added to neutralize some of the Tris base to form the buffering Tris acid/base pair.
The cells when disrupted should transfer soluble proteins into this buffer well and the added SDS is expected to denature and solubilize more proteins. But SDS precipitates in cold solutions and this could bring down some of your protein and hide them in the pellet so you need to look out for this. I'd make up fresh buffer and some RIPA buffer and try a very small pilot experiment to follow where your protein goes. Realize too that protein assays are often very sensitive to detergents like SDS, maybe look into detergent tolerant assays (BioRad) include SDS in your protein std etc to be sure you are measuring proteins accurately. You can always run two gels and coomasie stain one to see total protein and then be confident you loaded enough if the Western is weak. Good luck!
Edward,
Thank you for all of your help and tips!! It turned out our reagents A and B were bad- even though they were brand new. I appreciate your help!
Great! Its often best to make stable concentrated stocks of things like Tris, EDTA and NaCl and then make up small batches of the diluted buffer as needed. Once you know the stocks are good the diluted working buffers can be trouble free and highly reproducible.
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