Proceed with immunodetection (see Immunodetection using a chemiluminescent detection method or Immunodetection using a chromogenic detection method).Tip: For larger sample volumes, suitable equipment is available from several suppliers. NoteThe spotted membrane can be stored at 4C or at room temperature in a plastic bag until needed. Place the membrane on a UV trans-illuminator (spotted side down) and cross link the probe to the membrane for 1-5 minutes. After applying the samples, the membrane should be dried for a short time at room temperature before proceeding with the detection process. Background Fluorescence-based multiplex western blot detection is particularly suited for simultaneous detection of phosphorylated and total protein. Air-dry the spotted membrane at room temperature for 30-45 minutes or at 80C for 10 minutes.Tip: To differentiate between nonspecific and positive signals, an extra sample containing 1 µl of a cell extract of the host strain without plasmid (or other suitable control) should also be applied to the membrane and treated together with the protein of interest. In most cases diluting the protein with buffer containing denaturing reagents will increase epitope exposure and give better results. Dilute the nucleoside competitors serially in blocking buffer to give final concentrations of 1000/200/40/8/0 nM. Note: Under native conditions especially, the antibody epitope must be at least partially exposed to allow antibody binding. Wash the membrane in wash buffer for 10 min. Take mental note of where your blot is on the ruler on the scanning surface. Denature the diluted RNA sample at 95 degrees in a heat block to disrupt secondary structures for 3 min.
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Cover with clear rubber mat and roll with roller to remove any air pockets. Procedure Dilute the RNA samples or oligos containing the modification of interest to an appropriate concentration with RNase free water. It is also possible to use crude cell lysate and apply 1 µl samples with an estimated concentration of 1–100 ng/µl protein. Place blot (wet membrane if scanning for total protein, dry membrane if scanning finished blot) on scanner, protein side down. Apply 1 µl samples of diluted protein directly onto membrane.You get your solubilized protein in the supernatant. Tip: The protein of interest is diluted in dilution buffer for denaturing conditions, dilution buffer for native conditions, or another preferred buffer. Put the sample in cold ice and leave the sample during one hour and after that you can speed 45.000 x g during 1 Hour. Dilute protein samples in buffer to final protein concentrations of 1–100 ng/µl.
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