Anti-Src (phospho Y418) antibody (ab4816)

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 (1:2500) -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 (1:2500) -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

Cells were washed twice in PBS, lysed in RIPA lysis buffer (Tris–HCl pH 8.0 25 mM, NaCl 150 mM, NP40 1%, sodium deoxycholate 1%, SDS 1%, Na3VO4 1 mM, EDTA 0.5 M, and complete protease and phosphatase inhibitor cocktail 2 mM) and subjected to a 3x sonication burst cycle for 5 seconds at 30-40%. The cells were ultimately pelleted for 30 seconds at 14000× g. After centrifugation, supernatant protein extracts were aliquoted and stored at −80°C until use. Protein levels were determined by Bradford assay using BSA (bovine serum albumin) as a standard. The appropriate protein quantity was dissolved in Laemli buffer (Tris–HCl, pH 6.8, 62.5 mM, glycerol 10%, SDS 1%, 2-mercapto ethanol 5%, bromphenol blue 0.0025%), and the proteins were separated on SDS-PAGE gels (12%) before they were blotted onto a nitrocellulose transfer membrane (Whatman - Protrans). The primary antibodies included the following: anti-Src antibody 1:2500 [EG107] – (Abcam ab32102), anti-Src (phospho Y419) antibody – (Abcam ab4816), p44/p42 MAPK (Erk1/2) Rabbit mAb 1:1000 (Cell Signaling 137F5), and tubulin 1:10000 (Sigma – T6557). The secondary antibodies included goat anti-rabbit Alexa Fluor 680 1:5000 (Invitrogen – A21057) and donkey anti-mouse IRDye 800CW 1:5000 (Rockland Inc. – 605-731-002). To inhibit c-Src, an inhibitor of Src family kinases, PP2 (Sigma #P0042-5MG), was supplied at a 10 μM concentration overnight before protein extraction.

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Manufacturer protocol

Download the product protocol from Abcam for Anti-Src (phospho Y418) antibody (ab4816) below.

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