Western transfers are an important tool for proteomics research, diagnostics, and forensics applications. They are widely used to detect the presence of proteins in unknown or complex sample mixtures, as well as determine the relative abundance of specific proteins. Our family of PVDF membranes are most commonly used for western transfers and have been optimized for compatibility with a wide range of protein stains and enzyme-based detection systems, specifically with chemiluminescent substrates. The physical characteristics of PVDF membrane, such as durability during stripping and reprobing and high tensile strength for ease of handling, make it an excellent choice for protein detection.
The need to detect and quantitate minute quantities of proteins in a sample requires the membrane to exhibit high signal-to-noise ratios with low background levels. Increasingly, the ability to detect multiple protein samples in a multi-plex modality is becoming an important aspect of protein detection. Autofluorescence from standard western transfer membranes can obscure specific signals, especially at shorter wavelengths. Pall’s FluoroTrans® PVDF membrane exhibits a markedly lower fluorescent background than standard PVDF membranes for Western transfers. This performance characteristic helps minimize the concern over autofluorescent signals from the solid phase media masking the detection signal of the protein. The sensitive nature of our membranes yield high resolution with low background and burn through with very little lot-to-lot variability. The very high levels of protein binding, as well as higher tensile strength and chemical compatibility than nitrocellulose, make our PVDF membranes the ideal choice for protein detection.
The FluoroTrans name is a trademark of Pall Corporation and is not available for use.
Multiplex Western Transfer with Alexa Fluor 488/APC Fluorescent Detection
Plasma samples spiked when varied levels of myoglobin and troponin I were added to lanes on a NuPage 12% gel. After electrophoresis and transfer, membranes were blocked with casein and incubated with a mixture of rabbit anti-troponin and goat anti-myoglobin, followed by a cocktail of donkey anti-goat Alexa Fluor 488 and donkey anti-rabbit APC. Membrane was scanned immediately following the conjugate step using 450 nm and 635 nm excitation.
Sensitivity is 3-10 ng per lane for both antigens, similar to that obtained for the singleplex assays. Washing after the conjugate step did not improve results.
Achieve Greater Sensitivity in Western Transfers
Serial dilutions of E. coli lysates were transferred from a 10 to 20% gradient gel to Pall BioTrace PVDF and a competitive PVDF membrane, then probed with rabbit anti-E. coli antibodies. Proteins were visualized using peroxidase-conjugated goat anti-rabbit antibodies and 4-chloro-1-naphthol substrate solution.
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