![]() If using labeled secondary antibody conjugates, continue to step 10. If using fluorescently labeled primary antibodies, continue to step 11. Use a generous amount of wash buffer so blots move freely during washes. Rinse membrane three times with PBS or TBS with 0.1% Tween®-20, then wash 5x for 5-10 min.If using fluorescently labeled primary antibodies, protect from light. Incubate membrane with gentle rocking for 1-2 hours at room temperature or overnight at 4☌.Pour off the blocking buffer and add enough diluted antibody solution to allow the membrane to move freely with no stationary bubbles or dry spots. Dilute primary antibody to recommended concentration in fresh blocking buffer.to 1 hour at room temperature with gentle rocking. Use enough buffer to completely cover the blot and allow it to move freely in the dish. Place blot in a clean dish containing blocking buffer of your choice.For nitrocellulose membranes, proceed to step 5. If using PVDF membranes, re-wet the membrane in methanol, then rinse in water.Our Mix-n-Stain™ Total Protein Prestain Kit can detect as little as 1 ng total protein per lane. Note: Ponceau S can be used for visual staining of cell lysate proteins at ~10 ug total protein per lane, but may not be sensitive enough to detect lower protein loading amounts. Optional: Confirm protein transfer by imaging total protein prestain (if used), or by staining the membrane with Ponceau S dye according to the supplier instructions.Note: After transfer, membranes can be rinsed in water, dried, and stored between sheets of filter paper at room temperature for months or longer. Perform SDS-PAGE and western transfer using standard protocols.Optional: To fluorescently label total protein in your sample for transfer confirmation and western normalization, use a total protein prestaining kit, such as our Mix-n-Stain™ Total Protein Prestain Kit, according the kit protocol.The blot should move freely in the buffer. For blocking and wash steps, don’t skimp on volume.Enough solution should be used to freely move across the blot without trapping bubbles. It may be desirable to minimize the volume of antibody solutions used for blotting by using sealable bags or small containers.Commercially available blocking buffers developed specifically for fluorescent western detection, such as our TrueBlack® WB Blocking Buffer, can give lower background than other blocking agents. These blocking agents are usually used at 1-5% in PBS (or TBS) + 0.1% Tween®20. ![]() BSA, non-fat dry milk, and fish gelatin can be used for western blot blocking and antibody dilution buffers.Some researchers prefer to use TBS for phosphoprotein detection out of concern that phosphate buffers may interfere with phospho-specific antibody binding. In our experience, PBS and TBS can be used for routine fluorescent western detection with similar results. At that time, Tris-buffered saline (TBS) was the buffer of choice for western blots, because phosphate buffers could interfere with alkaline phosphatase signal development. Before the development of chemiluminescence-based and fluorescence-based western detection, alkaline phosphatase substrates were commonly used for western detection. ![]() Alternatively, commercially available black blotting boxes for fluorescent westerns come in a variety of sizes for blots or membrane strips. 9 cm 2 petri dishes hold 5-10 mL and are convenient for washing and incubating mini-blots.After protein transfer, dried blotting membranes can be stored at room temperature for months to years prior to detection.In our experience, nitrocellulose and low fluorescence PVDF membranes show similar background fluorescence, but PVDF can give higher sensitivity, possibly due to higher protein binding. Either nitrocellulose or PVDF may be used for fluorescent western, but autofluorescence can vary widely among different sources of blotting membrane.Blue tracking dyes in SDS-PAGE loading buffer can fluoresce in the far-red/near-infrared spectra loading buffer with an orange tracking dye is recommended for fluorescent western detection.Optimal protein loading amount varies depending on detection method and target expression level, but ranges between 1-10 ug/lane for most applications. ![]() Visible fluorescent dyes can be used, but generally will have lower signal-to-noise ratio due to higher autofluorescence of proteins and blotting membranes in the visible spectrum.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |