Western blot

Western blotting transfer and detection procedure

March 2007

CONTENT

I. II. IV. V. VI.

REQUIRED REAGENTS AND EQUIPMENT SAMPLE PREPARATION ELECTROBLOTTING AND BLOCKING IMMUNODETECTION BUFFER RECIPES

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5 7 8 9 10 11 12 13

III. ACRYLAMIDE GEL PREPARATION AND ELECTROPHORESIS

VII. OPTIMIZATION STEPS AND GENERAL TIPS VIII. TROUBLESHOOTING GUIDE IX. X. FLOW CHART NOTES

2I. REQUIRED REAGENTS AND EQUIPMENT Reagents: PVDF membrane e.g. 0.45μm Immobilon-P (Millipore IPVH00010) Blocking buffer – 5% fat free milk in PBS Phosphate buffered saline, PBS (recipe see section VI) MitoSciences’ primary monoclonal antibody(ies) Secondary anti-mouse antibody (typically goat anti mouse) which should be conjugated appropriately for the detection method of choice (see sectionV) (MitoSciences MS901 - MS908) Tween-20 (Aldrich 27,434-8) Double distilled water Methanol CAPS (Sigma C2632)

Equipment: Vertical acrylamide electrophoresis unit-BioRad mini Protean series recommended Electroblotting unit-fully submerged BioRad mini Protean series recommended pH meter, weighing balance and other standard lab equipment

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II. SAMPLE PREPARATION It is always recommended toisolate mitochondria from cells before analysis. Protocols for mitochondrial isolation can be found at www.mitosciences.com/PDF/mitos.pdf It is possible to probe whole tissue or cell extract but this may result in a weaker signal and/or additional bands resulting from non-specific cross-reactivity to non-mitochondrial proteins. In our experience such non-specific cross-reactivity is limited.Samples should be solubilized in the SDS-PAGE loading buffer detailed in section VI. After solubilization the sample should be spun at 13000 rpm for 5 minutes. The supernatant should be collected and loaded onto the gel. In most cases the sample need not be heated/boiled in this sample buffer prior to loading. However sample heating may be incorporated into this protocol if necessary, see section VIII.MitoSciences’ antibodies have been optimized for the detection of mitochondrial proteins over a wide range of antigen concentrations however suggested concentrations are presented in Table 1 providing the user with the opportunity of producing the best signal. Using these suggested sample amounts should yield a linearly proportional signal allowing quantitation.
Table 1. Suggested proteinloading concentrations. Using these loading amounts as simple guidelines in conjunction with the recommended electrophoresis, transfer, and immunodetection methods detailed in this manual should yield optimal, reproducible and quantitative Western blotting results with MitoSciences monoclonal antibodies.

Sample Heart mitochondria Muscle mitochondria Brain mitochondria Other tissue mitochondria Muscletissue extract Non-muscle tissue extract Cultured cell mitochondria Cultured cell extract

Loading 1-10 μg/lane 2-10 μg/lane 5-20 μg/lane 5-20 μg/lane 3-30 μg/lane 20-50 μg/lane 10-20 μg/lane 20-50 μg/lane

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III. ACRYLAMIDE GEL PREPARATION AND ELECTROPHORESIS Most commercially available pre-cast gel systems are suitable e.g. Invitrogen NOVEX NuPAGE and BioRad Ready gel/Criterion systems.Electrophoresis using pre-cast systems such as these should always be performed according to the manufacturer’s instructions. Alternatively acrylamide gels can be poured by hand. While it is possible to use a single acrylamide concentration such as a straight 15% gel, we highly recommend the use of a linear acrylamide concentration such as 10-20% which will give optimal resolution of all proteinsbetween 10 kDa and 100 kDa. A recipe for pouring 10-20 % acrylamide gels in the a 10x gel BioRad Mini-PROTEAN II multicasting chamber is detailed in Table 2 when using a two chamber gradient mixer.

BioRad MiniProtean II gel

Typical gradient former casting chamber (165-2950)

Table 2. Recommended acrylamide – BioRad 30% Acrylamide/Bis Solution 37.5:1 (161-0158).

10% acrylamide 10.6 ml...