Anti-TNF alpha antibody (ab6671)

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

IA tissue samples were prepared and treated with 3 ml lysate containing 7 mol/l carbamide, 2 mol/l thiocarbamide, 5 ml/l IPG buffer (pH 3–10), 65 mmol/l dl-DTT, 40 g/l CHAPS, 5 mg/l protease inhibitor, and 10 ml/l trypsin inhibitor. Then the tissues were subjected to phacofragmentation on ice. Next, the samples were centrifuged at 120000 g for 30 min at 4°C. The obtained supernatant was protein extracts. BCA method was used to determine the protein concentration. The protein was inactivated with 5× serine dehydratase (SDS) lysate (P0013G, Beyotime, Beijing, China) at 100°C for 5 min, followed by electrophoresis on polyacrylamide gel (5% concentration gel and 12% separation gel) with 20 μl sample uploaded. After transfer of membrane, TBS with Tween 20 (TBST) containing 5% BSA was blocked with decolorization table at room temperature for 1 h. Then sealing liquid was aspirated, the membrane was placed into plastic groove with the addition of following primary antibodies: rabbit anti-Apc polyclonal antibody (containing 5% BSA, ab15270, 1:2000), rabbit anti-NF-κB p65 polyclonal antibody (ab19870, 2.5 µg/ml), rabbit anti-IκBα polyclonal antibody (ab7217, 1:2000), rabbit anti-MMP-2 polyclonal antibody (ab97290, 1:2000), rabbit anti-MMP-9 polyclonal antibody (ab138306, 1:500), rabbit anti-TNF-α polyclonal antibody (ab6671, 1:1000), rabbit anti-IL-1β polyclonal antibody (ab9722, 0.2 µg/ml), rabbit anti-IL-6 polyclonal antibody (ab6672, 1:1000), and rabbit anti-p-p65 polyclonal antibody (ab86299, 1:1000) shaken and placed in a freezer at 4°C overnight. All these antibodies were purchased from Abcam Inc. (Cambridge, MA, U.S.A.). On the following day, the membrane was washed with TBST for three times (10 min/time), and added with diluted secondary antibody goat anti-rabbit in the same way (ab6721, Abcam Inc., Cambridge, MA, U.S.A.), incubated for 4–6 h at 4°C, and then the membrane was washed three times with TBST (15 min/time). Electrochemiluminescence of tris (2,2′-bipyridine) ruthenium (II)/tri-n-propylamine (TPA) (Yanhui Biotech Itd., Shanghai, China) were mixed at a ratio of 1:1, dropped off to the nitrocellulose (NC) membrane and developed with. Relative OD value was analyzed for all immunoblotting bands. The experiment was repeated three times to obtain the mean value.

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Papers

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Manufacturer protocol

Download the product protocol from Abcam for Anti-TNF alpha antibody (ab6671) below.

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Videos

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