Cells were lysed in cell lysis buffer (cell signaling technology, USA) with a protease inhibitor cocktail (BBI Life Sciences, China). Proteins were separated by 10% or 12% SDS-PAGE and transferred onto PVDF membranes. After blocking in 5% non-fat milk, the membranes were incubated with primary antibodies overnight at 4°C and detected by incubating with specific secondary antibodies (Jackson ImmunoResearch, USA) for 2 h at room temperature. Then protein bands were exposed with ECL chromogenic substrate. β-Actin (Huaan biotech, China) served as an internal loading control. Other primary anti-human antibodies include UCH-L5 (Santa Cruz, USA), SNRPF (abcam, USA), Flag-tag (MultiSciences Biotech, China) and HA-tag (Abmart, China).
Specific secondary antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
Specific secondary antibodies are laboratory reagents used to detect and label primary antibodies in immunoassays and other immunodetection techniques. They are designed to bind to the Fc region of primary antibodies, allowing for the visualization and amplification of the primary antibody signal.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ha tag, by Abmart (1 mentions)
Snrpf, by Abcam (1 mentions)
Flag tag (flag), by MultiSciences Biotech (1 mentions)
Protease inhibitor cocktail, by BBI Lifesciences (1 mentions)
Tris acetate gel, by Thermo Fisher Scientific (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Anti glt 1, by Alpha Diagnostic (1 mentions)
Anti vimentin, by Merck Group (1 mentions)
Anti gfap, by Agilent Technologies (1 mentions)
Inverted microscope, by Nikon (1 mentions)
T jnk, by Santa Cruz Biotechnology (1 mentions)
T erk1 2, by Santa Cruz Biotechnology (1 mentions)
P jnk, by Santa Cruz Biotechnology (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Protein lysis buffer, by iNtRON Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Imagequant las 4000, by Fujifilm (1 mentions)
4 protocols using specific secondary antibody
1
Western Blot Analysis of Protein Samples
2
Western Blot Analysis of Protein Samples
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Protein concentration was measured using a Pierce BCA protein assay kit (Thermo Scientific). Subsequently, 10–50 μg of protein was loaded onto 4–12% Bis–Tris gels (Thermo Scientific) and transferred to polyvinylidene difluoride (PVDF) membranes for 1.30 h. The membranes were incubated in 5% BSA or milk for 1 h at room temperature and incubated overnight at 4 °C with the appropriate primary antibodies. Individual antibodies used are listed in suppl. Table 2. The next day, the blots were washed three times with TBS-T for 10 min and incubated with the specific secondary antibodies (1:20,000, Jackson laboratory) for 1 h at RT. The blots were then washed with TBS-T, and imaged/quantified using a Syngene G:Box. For the detection of MLKL monomers and dimers, proteins were run on 3–8% Tris–acetate gels (Thermo Scientific) in non-reducing conditions.
3
Immunocytochemistry Protocol for Cell Culture Analysis
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Immunocytochemistry was performed as previously described23 (link). Briefly, cell cultures were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature. After blocking overnight with 4% albumin, the cells were incubated overnight with anti-GFAP (1:400; Dako [Z0334], Carpienteria, CA), anti-GLT-1 (1:1000; Alpha diagnostic [GLT11-A], San Antonio, TX), anti-vimentin (1:1000; Sigma Aldrich [V6630], St. Louis, MO) or anti-NeuN (1:50; EDM Millipore [MAB377], Billerica, MA) at 4°C, followed by PBS washes and incubation with a specific secondary antibody (Jackson ImmunoResearch, West Grove, PA) conjugated with Alexa Fluor® 488 (green staining, [111-545-003]) or 594 (red staining, [315-585-003]) for 1 h at room temperature. For all the immunostaining-negative controls, reactions were performed omitting the primary antibody. No reactivity was observed when the primary antibody was excluded. Cell nuclei were stained with 0.2 mg/mL of 4′,6′-diamidino-2-phenylindole (DAPI; EDM Millipore [268298], Billerica, MA). The cells were visualized with a Nikon inverted microscope and the images were transferred to a computer with a digital camera (Sound Vision Inc.).
4
Western Blot Analysis of Signaling Proteins
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Total cell lysates were prepared with protein lysis buffer (Intron Biotechnology, Seoul, Korea) supplemented with 1× protease inhibitor cocktails and PMSF (Sigma-Aldrich, St. Louis, MO, USA). The lysates were suspended with sample buffer containing sodium dodecyl sulfate (SDS) and boiled for 7 min for denaturation. Proteins were separated on 8% or 12% SDS-polyacrylamide gels electrophoresis, and then transferred onto a polyvinylidene fluoride (PVDF) membranes. The membrane was incubated with primary antibodies in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) for overnight at 4 °C. Primary antibodies for p-ERK1/2, t-ERK1/2, p38, p-JNK, t-JNK, AKT, and β-actin were purchased from Santa Cruz biotechnology Inc. (Santa Cruz, CA, USA). p-p38 and p-AKT antibodies were obtained from (Cell Signaling Technology Inc., Danvers, MA, USA). The membrane washed with TBS-T to remove primary antibody, followed by incubation with specific secondary antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) for 2 h. The signals were visualized using enhanced chemiluminescence (ECL; Abclon, Seoul, Korea) and an Image Quant LAS-4000 (Fujifilm Life science, Tokyo, Japan).
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