Dilute the secondary antibody 1:10.000 in blocking buffer or according to the manufacturers’ instructions.Incubate at 4˚C for 1 h on an end-over-end shaker.Note: To reduce the amount of antibody solution required, the membrane can be sealed in a plastic bag. Note: Save the supernatant for SDS-PAGE analysis to check for successful binding of the bait protein. Place the tube again on the magnetic microtube stand and allow the beads to separate. Add 250 µL Buffer BW and mix by vortexing. Note: Depending on the antibody, other dilutions of primary and secondary antibodies might be required, e.g. Dilute Penta-His antibody 1:1000 in blocking buffer and incubate the membrane in the diluted antibody solution for 1 h.Wash twice for 10 min each with 10 mL TBS-TT buffer.Place the tube on a magnetic microtube stand until the beads are separated and discard the supernatant. Add 250 µl of BW buffer and mix by vortexing.Note: Perform all incubation steps at room temperature (15-25☌) Wash membrane twice for 10 min each time with 10 mL TBS buffer. Place membrane in a 50 mL Falcon tube or a suitably sized plastic box and place it on a shaker.Remove excess staining solution with double distilled water and check for successful protein transfer. Dismantle the western blot sandwich and stain the membrane with Ponceau solution.To ensure that you are not losing any protein, put two membranes on top of each other. blotting time for a 30 kDa protein would be 35-40 min). Allow 1 min per kDa of protein and add 5-10 min (e.g. Note: It is important to adjust the blotting time to the protein of interest. Run the western blot at 400 mA constant electric current for 30-60 min. Place a heavy weight on the blot chamber.Alternatively, PVDF membranes can be used. 3 layers blotting paper Note: Most proteins, including membrane proteins, blot well on nitrocellulose membranes. Set up a western blot sandwich in a semi-dry blotter as follows:.Instructions: Mix all components and fill volume up to 50 mL using double distilled water.ĬL 2 (Chemiluminescence Detection Solution 2) (50 mL) Alternatively, take 200 ml TBS buffer and add Tween 20 and Triton X-100.ĬL 1 (Chemiluminescence Detection Solution 1) (50 mL)Ġ.15 g in 10 mL DMSO Store as 220 µL aliquot at -20☌Ġ.44 g in 10 mL DMSO Store as 500 µL aliquot at -20☌ Instructions: Mix all components and fill volume up to 500 mL using double distilled water. Instructions: Dissolve 3 g of milk powder in 100 ml TBS buffer. Instructions: Mix all components and fill volume up to 500 mL using double distilled waterīlocking Buffer (100 mL,3% milk Powder (w/v)) Instructions: Mix all components and fill volume up to 100 mL using double distilled water. Set pH to 8.0 using HCl and fill up to 1000 mL. Instructions: Mix all components and fill up volume to 900 mL with double distilled water. Composition of Solutions and buffers Western Blot Transfer Buffer (1000 mL)
#Western blot protocol for free
All our protocols are available for free download on our Protocols & datasheets page. Please refer to the dedicated protocols for detection of Rho1D4-tagged and GST-tagged proteins. Other antibodies might require different buffers, antibody dilutions, and incubation times. Note that this protocol was optimized for this particular antibody. The PentaHis antibody specifically recognizes the epitope HHHHH. This protocol describes the blotting of proteins from an SDS-PAGE gel onto western blot membranes, and the subsequent detection of his-tagged proteins using the Cube PentaHis antibody and chemoluminescence detection reagents.
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