Run the gel in MES SDS running buffer at 200V for 35 min. Load the histone samples, including a pre-stained protein standard with sufficiently low molecular weight markers. A higher percentage gel (15%) is recommended for a more effective resolution of histones.ģ. Prepare a 10% Bis-Tris gel of 1.0 mm thickness. Please note that protein loadings derived from cellular lysates will need to be determined empirically.Ģ. Centrifuge the sample briefly to restore sample volume from condensation formed in the tube during heating. Ělways use loading control antibodies to standardize your experiments.ġ.ğor each lane, prepare 0.5 µg calf thymus or acid extracted histones diluted in 1X LDS sample buffer, supplemented with 100 mM DTT.However, the optimal blocking is antibody dependent and should be empirically determined. Using high-quality BSA in your blocking solutions is recommended over dried milk powders.Use a nitrocellulose membrane with a pore size of 0.2 µm to ensure the optimal capture of histone proteins.Use a high-percentage gel for a clear resolution of histone proteins.Tips for successful western blotting with histones: Histones have a very low molecular weight, usually around 10–20 kDa, and due to their small size, you need to be careful when carrying out your western blot. Rinse the blot in TBST 3–4 times for 5 minutes each at RT. Note that 1/5000 is usually a good working dilution although this needs to be optimized for the particular application.ġ0. Rinse the blot in TBST 3–4 times for 5 minutes each at room temperature (RT).ĩ.ĝilute the horseradish peroxidase (HRP) labeled secondary antibody at the recommended dilution in TBST. Incubate overnight at 4☌ with agitation.Ĩ. We recommend incubating in a sealed bag, hybridization tube, or 50 ml Falcon tubes (~2.5 ml primary antibody/blot). Incubate for 1 h at 4☌ with agitation.ħ.ĝilute the primary antibody in TBST to the recommended dilution. We recommend not washing blots in distilled water as this can strip off proteins in some circumstances.Ħ.ělock the membrane with 5% w/v BSA in TBST. The stain can be removed by washing in PBST or TBST. If required, the transfer efficiency can be determined by staining the membrane briefly (10 s) in Ponceau stain. If using PVDF, note that it is essential to activate the membranes by pre-wetting in 100% methanol for 10 seconds before transfer.ĥ. Transfer the proteins to the membrane using semi-dry or wet transfer methods. Load the sample onto an SDS-PAGE gel and run the gel under standard conditions.Ĥ. For non-reduced samples, don't add DTT or ß-mercaptoethanol.Ģ.ĝenature the proteins by heating the sample to 95☌, or boiling, for 5 min.ģ. For reduced samples, the sample buffer should be supplemented with DTT or ß-mercaptoethanol. To a sample of protein solution containing 1–100 ng of the target protein (500 µg lysate), add an equal volume of 2x SDS-PAGE sample buffer. Western blot protocol for phosphorylated proteins:ġ. A starting BSA concentration of 5% w/v in TBS-T is suggested, however, the optimal concentration is antibody dependent and should be empirically determined. Casein is a phosphoprotein, present in milk, which causes high background signals. It is also important to block the membrane in BSA and avoid using casein or milk blocking buffers. To keep the proteins in their phosphorylated state, add phosphatase inhibitors to the working stocks of your buffers and always keep samples on ice. If you’re working with post-translational modifications of proteins, such as phosphorylation, it’s essential to handle your samples with care and use the right reagents to maintain the structural integrity of your proteins. In the videos below, we cover all common causes of no signal, high background, and multiple bands. If you are having difficulties getting your western blots exactly how you want them, take a look over our troubleshooting tips. We’ll also explain how to detect histones and phosphorylated proteins.ģ.3 Western blot protocol for phosphorylated proteinsĪll experiments and model systems are different, so it may not be enough to follow a standard protocol to get the results you are expecting. In Part 3 of our series on western blot, we’ll show you a few troubleshooting tips and answer frequently asked questions (FAQs). We’ll guide you through western blot basics and essential protocols before moving on to optimization, troubleshooting, and more advanced techniques. Welcome to our training series on western blot.
0 Comments
Leave a Reply. |