![]() It is not advisable to make quantitative comparisons of targets probed before and after stripping since the procedure removes some sample protein from the membrane. When probing for multiple targets, stripping and re-probing a single membrane instead of running and blotting multiple gels has the advantage of saving samples, materials, and time. Stripping is useful when one wants to investigate more than one protein on the same blot, for instance a protein of interest and a loading control. Stripping is the term used to describe the removal of primary and secondary antibodies from a western blot membrane. The legend at top indicates the specific conditions used for each column of cells.Our protocol for western blot membrane stripping and restaining includes step-by-step details on the removal of antibodies from western blot membranes.ĭownload the complete western blot guide. The dark regions in columns H3-6 is thought to be disruption of the cells by the pipette tips. Note that detection of tubulin is demonstrated only in the permeabilized cells (columns 3- 6). A 96-well tissue culture plate was seeded with Capan-2 cells, and after treatment with mouse anti-tubulin and Dylight™ 800 conjugated anti-mouse secondary, alpha-tubulin is detected. After the last wash, gently pipette out any residual liquid and blot plate dry.įigure: In-cell Western data showing detection of tubulin in permeabilized Capan-2 cells.Incubate with secondary antibody for 1 hour at room temperature with gentle shaking.Cover plate with aluminum foil to protect the Dylight™ from exposure to light. Add 50 µL/well Anti-MOUSE IgG (H&L) Antibody Dylight™ 800 diluted 1:1000 in blocking buffer.Wash by adding 200 µL/well 1X PBS + 0.1% Tween-20 4 times for 5 minutes each at room temperature with gentle shaking.Incubate primary antibody for 2 hours at room temperature with gentle shaking then move to 4☌ for overnight incubation without shaking.Add 50 µL /well mouse anti-alpha tubulin primary antibody diluted to 2.5 µg/mL (1:400) in blocking buffer.Wash by adding 200 µL /well 1X PBS + 0.1% Tween-20 4 times for 5 minutes each at room temperature with gentle shaking.Incubate blocking step for 1.5 hours at room temperature with gentle shaking.Block by adding 150 µL/well of 1X PBS fish gel solution.Periodically check to confirm cells have not detached from the plate. Gently flick out contents of wells between each wash.Wash and permeabilize cells with 200 µL/well of 1X PBS + 0.1% Triton X-100 4 times for 5 minutes each at room temperature with gentle shaking.Remove fixing solution by gentle aspiration using multichannel pipet.Incubate 20 minutes at room temperature without shaking. Add 150 µL/well 3.7% formaldehyde in 1X PBS.Remove media from wells and gently wash the cells 2 times with 1X PBS by adding 200 µL/well for 5 minutes each at room temperature with gentle shaking.Note: Permeabilization is not necessary for antibodies that target extracellular protein domains. As a negative control, cells in a portion of the plate were not permeabilized. Permeabilization with Triton X-100 allows for greater antibody penetration into the cell interior. Since alpha-tubulin is cytosolic, the cells are fixed and permeabilized. Allow the cells to grow to confluence (for Capan-2 cells approximately 48 hours). The recommended seeding density is 0.2 x 10 6 cells per mL. Black-walled plates may be used to reduce side scatter. The cells are trypsinized and then seeded into a 96-well tissue culture plate. In this experiment, Capan-2 pancreatic cancer cells are grown and seeded into a 96-well ELISA plate and probed for alpha-tubulin.Ĭapan-2 cells are grown to sub-confluence in a T-150 flask. The following example is a positive control experiment to demonstrate the detection of tubulin. Procedure for Tubulin Detection as a Positive Control
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