These plug-and-play antibodies allow simultaneous detection of up to three proteins (two targets of interest and one housekeeping protein) on the same blot when used with the hFAB Rhodamine Housekeeping Protein Fluorescent Primary Antibodies. To address these workflow challenges, Bio-Rad created the StarBright Blue line of secondary antibodies that enable highly sensitive fluorescent detection, short exposure times, and easy multiplexing for western blotting. An alternate method involves attempting to sequentially probe a single blot with multiple fluorescent antibodies to distinguish the signals from each one however, these protocols are cumbersome to optimize and execute. Scientists often resort to cutting or reprobing blots, which introduces variability and results in protein loss, thereby rendering the blot irreproducible and unsuitable for quantitation. Using traditional chemiluminescent methods to detect and quantify multiple proteins simultaneously can be challenging. They exhibit a two- to threefold lower limit of detection than traditional green emission fluorophore-labeled antibodies such as Alexa Fluor 488 or DyLight 488, the current industry standards. announced the launch of StarBright Blue 520 Fluorescent Secondary Antibodies, fluorescent dye–labeled secondary antibodies for use in multiplex western blotting. Mix the Clarity Western ECL Substrate Kit components in a 1:1 ratio.
It is important to use an ECL substrate that has good sensitivity and long signal duration, such as the Clarity Western ECL Substrate. After the final wash step, keep the blot in TBST while preparing for blot detectionĪll PrecisionAb Antibodies were validated using enhanced chemiluminescent (ECL) detection. Rinse the blot with 15 ml TBST at RT for 5 min. Incubate the blot in the secondary antibody and blocking buffer solution at RT for 1 hr with gentle agitation Please refer to the antibody product page for details on the exact secondary antibody used during the validation process. Repeat for a total of five washesĭilute the appropriate secondary antibody in 10 ml blocking buffer according to the following table: Incubate the blot in the primary antibody and blocking buffer solution at 4☌ overnight with gentle agitation Please see the validation protocol (bulletin 6603) for more details.ĭilute the primary antibody 1:1,000 in 10 ml blocking buffer If using BSA, you may notice some nonspecific bands due to its low stringency. We recommend using casein or nonfat dried milk for blocking. When using casein, do not block for longer than 30 min to prevent reduction in signal specificity. Load the control cell lysate adjacent to your samples and the molecular weight (MW) marker (see diagram).ĭuring the validation process, we blocked for 30 min at room temperature (RT) in blocking buffer + 0.1% Tween 20. If using BME, add 180 μl H20, 200 μl 2x Laemmli Sample Buffer, and 20 μl BME If using DTT, add 190 μl H20, 200 μl 2x Laemmli Sample Buffer, and 10 μl 2 M DTT Reconstitute 400 µg lysate in one of the following ways, depending on the reducing reagent used: Secondary antibodies (see antibody datasheet) Trans-Blot Turbo Mini PVDF Transfer Pack.Transfer membranes, reagents, and equipment 1x Tris/glycine/SDS (TGS running buffer).
#BIO RAD WESTERN BLOT ALL BLUE STANDARDS PLUS#
Precision Plus Protein All Blue Standards Value Pack.Mini-PROTEAN Tetra Cell for Mini Precast Gels.Any kD Mini-PROTEAN® TGX Stain-Free Precast Gels (10 well, 50 µl).4–15% Mini-PROTEAN® TGX Stain-Free Precast Gels (10 well, 50 µl).Phosphate Buffered Saline (PBS) containing 1% w/v BSA Reducing agents such as dithiothreitol (DTT) or ß-mercaptoethanol (BME)Įlectrophoresis gels, reagents, and equipment
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