Anti-Fas antibody (ab82419)

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 (1:1000) -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 (1:1000) -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

The liver tumors were homogenized into 10% (wt/vol) hypotonic buffer (pH 8.0, 1 mM EDTA, 5 µg/ml leupeptin, 25 mM Tris-HCl, 1 mM Pefabloc SC, 5 µg/ml soybean trypsin inhibitor, 50 µg/ml aprotinin, 4 mM benzamidine) to yield a homogenate. Primary antibodies were shown in Table I. The final supernatants from cells and tumors were then obtained by centrifugation at 14,000 × g for 20 min at 4°C. The protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with bovine serum albumin as a standard. Sample-loading buffer was added, the mixture was boiled for 5 min. And the total protein extract was used for western blot analysis. Total protein (40 µg) was loaded and proteins were separated using 10% SDS-PAGE and electrophoretically transferred to the polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were then blocked with 5% skim milk Tris-buffered saline with 0.1% Tween 20 (TBST), washed, and then incubated with primary antibodies overnight at 4°C. The membrane was then washed with TBST 3 times, followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:2,500; KeyGen Biotech, Nanjing, China) at room temperature for 2 h. Following another round of washing with TBST, the membrane was then developed using ECL (Thermo Fisher Scientific, Inc.), and exposed to Kodak X-ray film (Eastman Kodak Company, Rochester, Ny, USA). Every protein expression level was defined as grey value using ImageJ 1.38 software (National Institutes of Health, Bethesda, MD, USA) and standardized to housekeeping gene of GAPDH and expressed as a fold of control. All experiments were performed in triplicate and done three times independently.

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Manufacturer protocol

Download the product protocol from Abcam for Anti-Fas antibody (ab82419) below.

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