Protocol for PrecisionAb Western Blot Antibodies

Western Blot Protocol:
PrecisionAb™

PrecisionAb Antibodies have been extensively validated for western blotting. This protocol describes how to use PrecisionAb Antibodies to get the best western blotting results. At the end of the protocol, there are details for more resources that will provide comprehensive procedures and guidance to produce successful western blots.

Materials

Protein samples and reagents
  • Endogenous lysates
  • Reducing agents such as dithiothreitol (DTT) or ß-mercaptoethanol (BME)
Electrophoresis gels, reagents, and equipment
Transfer membranes, reagents, and equipment
Western blotting reagents and equipment
Imaging reagents and equipment

Method

  1. Lysate preparation

  • Reconstitute the 400 µg lysate in one of the following ways, depending on the reducing reagent used:
    • If using DTT, add 190 μl H20, 200 μl 2x Laemmli Sample Buffer, and 10 μl 2 M DTT
    • If using BME, add 180 μl H20, 200 μl 2x Laemmli Sample Buffer, and 20 μl BME
  • Heat at 95°C for 5 min.
  1. Gel Electrophoresis

  • Load the control cell lysate adjacent to your samples and the molecular weight (MW) marker (see diagram).
control cell lysates adjacent to samples and molecular weight marker
  • The amount of control lysate loaded is dependent on the gel comb size. Please see the recommendations in the tables below.
control lysate recommendations depending on gel comb size
  1. Blocking

  • During the validation process, we blocked for 30 min at room temperature (RT) in blocking buffer + 0.1% Tween 20. When using casein, do not block for longer than 30 min to prevent reduction in signal specificity. We recommend using casein or nonfat dried milk for blocking. If using BSA, you may notice some nonspecific bands due to its low stringency. Please see the validation protocol (bulletin 6603) for more details.
  1. Incubation with Primary Antibody

  • Dilute the primary antibody 1:1,000 in 10 ml blocking buffer
    • Incubate the blot in the primary antibody and blocking buffer solution at 4°C overnight with gentle agitation
    • Rinse the blot with 15 ml TBST at RT for 5 min. Repeat for a total of five washes
  1. Incubation with Secondary Antibody

  • Rinse the blot with 15 ml TBST at RT for 5 min. Repeat for a total of five washes
  • Dilute the appropriate secondary antibody in  10 ml blocking buffer according to the following table:
Primary Antibody Host Secondary Bio-Rad Catalog Recommended Dilution
Mouse GAM-HRP STAR207P 1:10,000
Rabbit GAR-HRP STAR208P 1:10,000
HuCAL Goat anti-human IgG F(ab')2:HRP STAR126P 1:2,500
Rat Goat anti-rat IgG:HRP STAR72 1:10,000
Goat Donkey anti-sheep/goat IgG:HRP STAR88P 1:10,000
Sheep Rabbit anti-sheep IgG:HRP 5184-2504 1:10,000
  • Please refer to the antibody product page for details on the exact secondary antibody used during the validation process.
    • Incubate the blot in the secondary antibody and blocking buffer solution at RT for 1 hr with gentle agitation
    • Rinse the blot with 15 ml TBST at RT for 5 min. Repeat for a total of five washes. After the final wash step, keep the blot in TBST while preparing for blot detection
  1. Blot Detection

  • All PrecisionAb Antibodies were validated using enhanced chemiluminescent (ECL) detection. It is important to use an ECL substrate that has good sensitivity and long signal duration, such as the Clarity Western ECL Substrate.
    • Mix the Clarity Western ECL Substrate Kit components in a 1:1 ratio. Prepare 0.1 ml solution per cm2 of membrane
    • Place the membrane, protein side down, in the substrate solution, and let the membrane develop for 5 min

Additional Resources

For more western blotting tips, please use the following resources

bulletin 2895
western blotting overview
troubleshoot western blot
 Bulletin 2895  Western blotting overview  Troubleshoot western blots

HuCAL is a trademark of MorphoSys AG.
Tween is a trademark of ICI Americas, Inc.
Precision Plus Protein Standards are sold under license from Life Technologies Corporation, Carlsbad, CA for use only by the buyer of the product. The buyer is not authorized to sell or resell this product or its components.