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Our Abpromise guarantee covers the use of ab56416 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.|
|WB||Use a concentration of 1 - 5 µg/ml.|
|ICC/IF||Use a concentration of 10 µg/ml. Fix cells for 15 minutes with 2 mL of 4% paraformaldehyde solution (pH 7.4 with NaOH in PBS). Permeabilize cells by incubating for 15 minutes on ice with 2 mL of 0.1% Triton X-100 in PBS.|
|Flow Cyt||Use 0.5µg for 106 cells.
ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.
|IHC-Fr||Use at an assay dependent concentration. PubMed: 22577215|
This image is courtesy of an Abreview submitted by Melanie Thelen.
Blocked with 5% milk for 3 hours at 21°C.
Incubated with the primary antibody for 17 hours at 4°C.
Image courtesy of Dr Randal Tibbetts by Abreview.All lanes are whole cell lysate prepared from HeLa cells. Treatments are listed.
Image from Vazquez-Martin A et al, PLoS One. 2009 Jul 16;4(7):e6251, Fig 4.Cells were washed twice with cold-PBS and then lysed in buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton® X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerolphosphate, 1 mM Na3VO4, 1 µg/mL leupeptin, 1 mM phenylmethylsulfonylfluoride, and complete protease inhibitor cocktail for 30 minutes on ice. The lysates were cleared by centrifugation in an Eppendorff tube (15 minutes at 14,000×g, 4°C). Protein content was determined against a standardized control using the Pierce Protein Assay Kit. Equal amounts of protein were resuspended in 5× Laemmli sample buffer (10 minutes at 70°C), resolved by electrophoresis on 10% SDS-PAGE, and transferred onto nitrocellulose membranes. Non-specific binding on the nitrocellulose filter paper was minimized by blocking for 1 hour at room temperature with TBS-T buffer [25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20] containing 5% (w/v) nonfat dry milk. The treated filters were washed in TBS-T and then incubated with the primary
This image is courtesy of an anonymous Abreview
Western blot analysis of Rhesus monkey retinal pigmented epithelium whole cell lysate (20µg/lane) treated with increasing concrentration of an autophagy inhibitor. SQSTM1 / p62 was labelling with ab56416 at 1/1000. An Alkaline Phosphatase-conjugated rabbit anti-mouse IgG polyclonal (1/1000) was used as the secondary antibody.