Skip to Content
Merck
  • Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS).

Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS).

PloS one (2015-08-20)
Jennifer E Gilda, Rajeshwary Ghosh, Jenice X Cheah, Toni M West, Sue C Bodine, Aldrin V Gomes
ABSTRACT

Western blotting is a commonly used technique in biological research. A major problem with Western blotting is not the method itself, but the use of poor quality antibodies as well as the use of different experimental conditions that affect the linearity and sensitivity of the Western blot. Investigation of some conditions that are commonly used and often modified in Western blotting, as well as some commercial antibodies, showed that published articles often fail to report critical parameters needed to reproduce the results. These parameters include the amount of protein loaded, the blocking solution and conditions used, the amount of primary and secondary antibodies used, the antibody incubation solutions, the detection method and the quantification method utilized. In the present study, comparison of ubiquitinated proteins in rat heart and liver samples showed different results depending on the antibody utilized. Validation of five commercial ubiquitin antibodies using purified ubiquitinated proteins, ubiquitin chains and free ubiquitin showed that these antibodies differ in their ability to detect free ubiquitin or ubiquitinated proteins. Investigating proteins modified with interferon-stimulated gene 15 (ISG15) in young and old rat hearts using six commercially available antibodies showed that most antibodies gave different semi-quantitative results, suggesting large variability among antibodies. Evidence showing the importance of the Western blot buffer and the concentration of antibody used is presented. Hence there is a critical need for comprehensive reporting of experimental conditions to improve the accuracy and reproducibility of Western blot analysis. A Western blotting minimal reporting standard (WBMRS) is suggested to improve the reproducibility of Western blot analysis.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Glycine, ACS reagent, ≥98.5%
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Tris(hydroxymethyl)aminomethane, ACS reagent, ≥99.8%
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Sodium dodecyl sulfate, BioUltra, for molecular biology, ≥99.0% (GC)
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Glycine, BioUltra, for molecular biology, ≥99.0% (NT)
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Sodium chloride, BioUltra, for molecular biology, ≥99.5% (AT)
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Sodium chloride solution, BioUltra, for molecular biology, ~5 M in H2O
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Sodium dodecyl sulfate, ACS reagent, ≥99.0%
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Glycine, 99%, FCC
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Trizma® base, BioUltra, for molecular biology, ≥99.8% (T)
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Urea-12C, 99.9 atom % 12C
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Sodium dodecyl sulfate, BioXtra, ≥99.0% (GC)
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Glycine, suitable for electrophoresis, ≥99%
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Trizma® base, Primary Standard and Buffer, ≥99.9% (titration), crystalline
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Anti-Mouse IgG (whole molecule)–Peroxidase antibody produced in rabbit, IgG fraction of antiserum, buffered aqueous solution
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Trizma® base, ≥99.9% (titration), crystalline
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Tromethamine, meets USP testing specifications
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Anti-Rabbit IgG (whole molecule)–Peroxidase antibody produced in goat, affinity isolated antibody
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Sodium chloride, BioXtra, ≥99.5% (AT)
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Sodium chloride, for molecular biology, DNase, RNase, and protease, none detected, ≥99% (titration)
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Sodium dodecyl sulfate, BioReagent, suitable for electrophoresis, for molecular biology, ≥98.5% (GC)
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Trizma® base, BioPerformance Certified, meets EP, USP testing specifications, suitable for cell culture, ≥99.9% (titration)
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Sodium dodecyl sulfate, ≥98.0% (GC)
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
DL-Dithiothreitol solution, BioUltra, for molecular biology, ~1 M in H2O
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Glycine, BioXtra, ≥99% (titration)
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Sodium chloride, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99%
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Sodium dodecyl sulfate, ReagentPlus®, ≥98.5% (GC)
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Glycine, ReagentPlus®, ≥99% (HPLC)
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Protease Inhibitor Cocktail powder, for general use, lyophilized powder
SKU
Pack Size
Availability
Price
Quantity
Sigma-Aldrich
Anti-Goat IgG (whole molecule)–Peroxidase antibody produced in rabbit, affinity isolated antibody, buffered aqueous solution
SKU
Pack Size
Availability
Price
Quantity
Supelco
Sodium dodecyl sulfate, dust-free pellets, suitable for electrophoresis, for molecular biology, ≥99.0% (GC)
SKU
Pack Size
Availability
Price
Quantity