Recombinant Anti-Claudin 1 antibody [EPR9306] (ab180158)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.	-Rabbit (1:50) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Upstream tips
These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.
Protocol tips
-Rabbit (1:50) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Publication protocol

For protein detection, Caco-2 cells were grown on 6-well transwell dishes as described in Section 2.2. At 21–23 days, cells were washed with ice-cold PBS, scraped and lysed in Bicine-CHAPS buffer (ProteinSimple, San Jose, CA, USA) containing 1% protease (P8340) and 1% phosphatase inhibitor cocktail (P0044 and P5762) (Sigma Aldrich). The lysate was centrifuged at 14,000g for 10 min at 4°C and the total protein concentration of the supernatant was determined by BCA microplate assay (Pierce Biotechnology, Thermo Fisher Scientific) according to manufacturer's instructions. MCT1, MCT4, ABCG2 and UGT1A protein abundance was determined using the ProteinSimple system “WES” (Bio-Techne, ProteinSimple) according to manufacturer's instructions. For MCT1, MCT4 and UGT1A detection, samples were denatured by incubation with DTT and SDS-containing Master Mix (Bio-Techne, ProteinSimple) at 37°C for 20 min. Because of differences in compatibility of antibodies when multiplexing and denaturing condition requirements for the individual protein targets, different loading controls were tested and selected for each protein of interest. Claudin 1, a tight junction marker (1:50, mouse monoclonal, #37-4900; Invitrogen, Life Technologies, Thermo Fisher Scientific) was used as a loading control and run in the same capillary as MCT1 (1:25, mouse monoclonal, sc-365501; Santa Cruz Biotechnology, Insight Biotechnology) and MCT4 (1:25, mouse monoclonal, sc-376139; Santa Cruz Biotechnology, Insight Biotechnology). For ABCG2 determination, α-tubulin (1:50, rabbit monoclonal, 2125S; Cell Signalling Technology, New England Biolabs, Hertfordshire, UK) was used as a loading control and detected from the same sample run in separate capillaries (1:50, rabbit polyclonal NBP1-59749; Novus Antibodies, Bio-Techne). Denaturation was carried out by incubation for 5 min at 95°C. Claudin 1 (1:50, rabbit monoclonal, ab180158; Abcam, Cambridge, UK) was also used as loading control for UGT1A (1:50, rabbit polyclonal, sc-25847; Santa Cruz Biotechnology, Insight Biotechnology) and both run in the same capillary. All antibodies were used in the linear response range following optimization (Fig. A.3). Quantification of peak areas and gel image reconstruction was carried out using the ProteinSimple Compass software. Every sample including the biotinylated ladder contained three fluorescent molecular weight standards that were used to align each individual capillary with the ladder and assign the molecular weights to detected peaks.

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Manufacturer protocol

Download the product protocol from Abcam for Recombinant Anti-Claudin 1 antibody [EPR9306] (ab180158) below.

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