R&D Systems Quality Control laboratories use these Western blotting and immunostaining protocols to show that our polyclonal and monoclonal antibodies are specific for the proteins they were raised against and to determine the sensitivity of the antibody for its antigen.
Protein samples are prepared with SDS and run under both reduced and non-reduced conditions on appropriate SDS-PAGE gel. The proteins are transferred to a PVDF membrane using a semi-dry transfer apparatus. For those proteins that have not been tested with natural samples, a protocol and troubleshooting guide is provided for Western blot optimization. Please note that for the antibodies that have been validated for natural samples, an optimized protocol is provided on the datasheet, which should be suitable for most samples.
|Western Blot Materials|
|Semi-dry Transfer Apparatus||Bio-Rad (Catalog # 170-3940) or equivalent|
|Power Supply||0 - 100 VDC (adj. current to 1 Amp)|
|PVDF Membrane||0.45 µm pore size; cut to same size as gel (Millipore, Catalog # IPVH304-FO) or equivalent|
|Filter Paper||Schleicher & Schuell 3MM or equivalent, cut to same size as gel|
|Wetting Solution||100% Methanol|
|Gel Running Buffer||19.3 mM Glycine, 2.5 mM Tris (Base), 0.1% SDS|
|Anode Buffer I||300 mM Tris, 20% methanol, pH 10.4|
|Anode Buffer II||25 mM Tris, 20% methanol, pH 10.4|
|Cathode Buffer||25 mM Tris, 20% methanol, 40 mM 6-aminocaproic acid, pH not adjusted|
|Sample Buffer||6% SDS, 0.25 M Tris, pH 6.8, 10% glycerol, 10 mM NaF and bromophenyl blue with or without 20 mM dithiothreitol (DTT)|
|Additional Tools||Forceps, clean plastic test tube, gloves, razor blade|
Note: Always wear gloves or use forceps when handling blotting membranes to avoid contamination with protein from fingers.
Bring all the solutions and reagents to room temperature before use; otherwise the detection limit may be compromised.
|Antibody Category||Working Concentration (microgram/mL)|
|Non Affinity Purified Polyclonal Antibody||1 - 2|
|Affinity Purified Polyclonal Antibody||0.1 – 0.2|
|Biotinylated Polyclonal Antibody||0.1 – 0.2|
|Monoclonal Antibody||1 – 2|
|Primary (1°) Antibody||Any polyclonal or monoclonal antibodies that have been validated for use in Western blots|
|Secondary (2°) Antibody||Depends on 1° antibody utilized, for example:
|Streptavidin Alkaline Phosphatase (SA-AP)||R&D Systems Catalog # AR001, or equivalent|
|Wash Solution (TTBS)||50 mM Tris, 0.5 M NaCl, 0.05% Tween 20, pH 7.4|
|Blocking Buffer||3% BSA (Fraction V, 3x crystallized), TTBS, 0.2% azide, pH 7.4|
|Primary and Secondary Antibody Solution||0.1% BSA in TTBS, pH 7.4|
|Diluent Buffer||1% BSA (Fraction V, 3x crystallized), TTBS, 0.2% azide, pH 7.4|
|AP Substrate Buffer||0.1 M Tris, 0.1 M NaCl, 5 mM MgCl2, pH 9.5|
|Color A||50 mg/mL nitro blue tetrazolium (NBT) in 70% dimethylformamide (DMF)|
|Color B||50 mg/mL bromochloroindolyl phosphate (BCIP) in 100% dimethylformamide (DMF)|
|Additional Tools||Forceps, clean plastic test tube, plastic trays, gloves, pipettes|