![]() ![]() Band loss and smearing can be seen at the higher loads for all targets on the Bio-Rad blot, while the blot for the NuPAGE Bis-Tris gel offers superior protein loading capacity above 24 µg and crisp, bright bands.įor immunodetection: membranes were blocked for 1 hour in 1X Blocker FL Fluorescent Blocking Buffer. Both membranes were probed for three targets (vinculin, α-tubulin, and p23) using fluorescent immunodetection. Western blots using NuPAGE 4-12% Bis-Tris midi gels generate sharper bands at greater protein and RIPA lysis buffer loads than Bio-Rad 4–20% TGX midi gels.A NuPAGE 4–12% Bis-Tris midi gel, 12+2 well, was loaded with decreasing total protein amount of HEK293 lysate, subjected to electrophoresis in a SureLock Tandem Midi Gel Tank and transferred onto a 0.45 µm PVDF membrane using the SureLock Tandem Blot Module and the Bio-Rad 4–20% TGX midi gel, 12+2 well, was subjected to electrophoresis in a Criterion Midi Cell Tank and transferred onto a 0.45 µm PVDF membrane using the Criterion Blotter. *Not all percentages are available in every well type WedgeWell format: 10, 12, 15 17 well (load up to 2X sample volume per well) SureLock Tandem Midi Gel Tank, Invitrogen XCell4 SureLock Midi-Cell or Bio-Rad Criterion (with adapters only) Mini Gel Tank or XCell SureLock Mini-Cell NuPAGE MES SDS or NuPAGE MOPS SDS Running Bufferīolt MES SDS or Bolt MOPS SDS running buffer Working with dilute samples or need to load larger sample volumes, or when faster run times are neededĬoefficient of variation (CV) of only 2% for Rf values (migration) ![]() This video demonstrates SDS-PAGE separation of proteins using the Bio-Rad Comparative Proteomics Kit II: Western Blot Module.Īssembly of the blotting sandwich and electroblotting are shown along with the steps for protein detection using a colorimetric assay.Need flexibility in well and gel format, or when you need a higher-throughput midi gel for a larger number of samples Western Blot Video: SDS-PAGE Separation of Proteins Please contact Bio-Rad’s Technical Services Department to learn about recommended secondary reagents for specific applications. It may be useful to include a sample in which no primary antibody is used at all, in order to determine any nonspecific binding of the secondary reagent to the target tissue. Wash the membrane with gentle agitation as follows: 4x 5 min in wash buffer 3x 5 min in PBST and 2x 5 min in PBS.Īdd appropriate enzyme substrate solution and incubate as recommended by the manufacturer to visualize protein bands.Īppropriate controls should always be carried out. Wash the blot extensively in wash buffer (3 x 10 min) with gentle agitation.Īdd appropriate enzyme-conjugated secondary antibody diluted in wash buffer and incubate for 1 hr at RT with gentle agitation. Incubate for 2 hr at RT, or overnight at 4☌. Rinse the blot briefly with wash buffer and then add primary antibody diluted in the wash buffer (a concentration of 1-10 µg/ml is generally acceptable, but check datasheets for precise recommendations). Place blot into blocking solution for 2 hr at RT, or overnight at 4☌. PBS Disodium potassium phosphate, 1.15g Distilled water, 1 L Potassium chloride, 0.2 g Potassium dihydrogen phosphate, 0.2g Sodium chloride, 8.0 g PBST Disodium potassium phosphate, 1.15 g Distilled water, 1 L Potassium chloride, 0.2 g Potassium dihydrogen phosphate, 0.2g Sodium chloride, 8.0 gįollowing SDS-PAGE, transfer proteins onto blotting membrane according to the manufacturer’s instructions.Ĭheck protein transfer by staining the blot with Ponceau S for 1 min, then completely destain the blot by washing with distilled water. Washing buffer Blocking buffer + 0.1% Tween 20 Ponceau S Acetic acid, 5 ml However, we advise using our protocol for detection of phosphorylated proteins by western blot. *Note: For cleaner western blots, Block Ace is recommended over 5% non-fat dried milk dissolved in PBS. For the detection of phosphorylated protein, use the recommended blocking solution as stated on the product datasheet. Blocking Buffer Block Ace BUF029 dissolved in water, or 5% non-fat dried milk dissolved in PBS. ![]()
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