Line up the developed film in the correct orientation to the blot and mark the bands of the MW ladder directly onto the film. Incubate the membrane completely with substrate for 1–5 minutes (adjust time for more sensitive ECL substrates, e.g., SuperSignal West Femto Chemiluminescent Substrate ).Įxpose the membrane to autoradiography film in a darkroom or read using a chemiluminescence imaging system. Prepare ECL substrate according to the manufacturer’s instructions. Tip: Do not let the membrane dry at any stage of the blotting process. Incubate for 1 hour with constant rocking. Incubate the membrane with a suitable HRP-conjugated secondary antibody (recognizing the host species of the primary antibody), diluted according to the instructions. Wash membrane three times with 1x TBST for 10 minutes each. (Optimal dilutions should be determined experimentally with a dilution series.) Incubate the membrane with primary antibody for 1 hour (or overnight at 4☌) on a benchtop rocker. If desired, stain the membrane with commercial Ponceau red solution for 30 seconds to visualize protein bands, then wash away Ponceau red solution with generous amounts of 1x TBST.īlock with 1x TBST containing (2–5%) non-fat dry milk (or 1–5% BSA for the detection of phospho-epitopes) with constant rocking for 1 hour or overnight at 4☌.ĭilute primary antibody in blocking solution with a starting dilution ratio of 1:1000. Gently mark the bands of the MW ladder on the membrane using a pencil. Therefore, the SDS bound proteins travel toward the positive electrode.įollowing transfer, wash the membrane twice with distilled water. The binding of SDS to proteins results in the complex having an overall negative charge. Use gel-loading pipette tips to facilitate easy and even loading of your protein samples.Ī representation of the components of a transfer “sandwich.” The PVDF membrane is situated nearest to the positive electrode. Apply semi-dry or wet transfer systems according to the instructions of the blotting apparatus manufacturer.Īfter removing gel combs rinse each well thoroughly with running buffer to ensure no remaining bits of acrylamide are blocking them. Sequentially assemble the transfer constituents according to the illustration shown on the next page (Figure 2), and ensure no bubbles lie between any of the layers. Soak the filter papers and sponges in transfer buffer. Soak membranes in methanol for 30 seconds before moving to transfer buffer. Please note: PVDF membranes (or PSQ membranes with 0.22um micropores for targets less than <30 kDa) are strongly recommended. Tip: Tris-tricine gels separate low MW proteins. Stop the gel running when the dye front migrates to the desired position. Turn on electrophoresis power pack and set to a low voltage (as the sample runs through the stacking gel), increasing to a higher voltage (e.g., 120V) when the dye front reaches the separating layer. Load samples and appropriate protein markers onto the gel using a tip. Remove gel combs and cleanse wells of any residual stacking gel by pipetting running buffer up and down in each well using gel-loading tip (Figure 1). Set up electrophoresis apparatus and immerse in 1X running buffer. Add 4X SDS sample buffer so the total protein amount is 30 - 50ug per sample (according to the protein amount measured by Bradford or BCA protein assay).įlick microfuge tubes to mix samples, spin them shortly, and then heat to 95 - 100 ℃ for 5 minutes. (For recipes see the "SDS-PAGE gel recipes" section) Construct an SDS-PAGE gel according to the molecular weight (MW) of your target protein(s).