Cell lysates were prepared by directly adding Laemmli buffer (SDS PAGE loading dye) into the culture plate, harvesting the cells and heating the sample at 95°C for 5 min. Samples were resolved on SDS-PAGE, followed by transfer of proteins on to polyvinylidene difluoride (PVDF) membrane (Millipore). Blots were blocked with 5% skimmed milk followed by incubation in primary antibody for 1 hr at room temperature or at 4°C overnight, washed and incubated with secondary HRP conjugated antibodies for 1 hr at room temperature, and washed extensively. The antibody dilutions used for immunoblotting were: LIC1–1:500, LIC2–1:500, Mad1–1:500, Mad2–1:250, β-actin- 1:2000, IC74–1:1000, myc—1:2000, anti-mouse HRP 1:10000, anti-rabbit HRP- 1:10000. The chemiluminescence signal was developed using the Luminata Forte reagent (Millipore) and captured in the Image Quant 4000 (GE).
Luminata forte reagent
Manufactured by Merck Group
Sourced in United States
Luminata Forte reagent is a laboratory reagent developed by Merck Group. It is designed to facilitate the detection and quantification of various biomolecules, such as proteins, in scientific research and diagnostic applications. The core function of Luminata Forte reagent is to generate a luminescent signal that can be measured using specialized laboratory equipment.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant, by GE Healthcare (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Trans blot turbo apparatus, by Bio-Rad (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Halt protease, by Thermo Fisher Scientific (1 mentions)
Dot blot apparatus, by Bio-Rad (1 mentions)
Superose 6 10 300 gl, by GE Healthcare (1 mentions)
Superdex200 10 30, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Imagequant las4000 gel documentation system, by GE Healthcare (1 mentions)
Las 4000, by Cytiva (1 mentions)
4 protocols using luminata forte reagent
1
Immunoblotting for Protein Expression
2
Biotin Switch Assay for Protein S-Nitrosylation
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The protein pellet after biotin switch assay was resuspended in HENS buffer (250 μl per mg of protein) and mixed with 3 volume of neutralization buffer (25 mM Hepes, pH 7.5, 100 mM NaCl, 1 mM EDTA and 0.5% Triton X-100). Streptavidin agarose beads were added to the protein solution (50 μl of streptavidin agarose beads per mg of protein) and incubated for overnight at 4 °C. Beads were washed five times with washing buffer (25 mM Hepes, pH 7.5, 600 mM NaCl, 1 mM EDTA and 0.5% Triton X-100) and protein was eluted with 20 μl of elution buffer (20 mM Hepes, pH 7.5, 1 mM EDTA, 100 mM NaCl and 2-mercaptoethanol). The eluted protein was mixed with equal volume of 2X SDS-PAGE sample buffer and was loaded onto 15% SDS-PAGE. The protein was transferred to PVDF membrane using Trans-Blot turbo apparatus (Bio-Rad, USA). The PVDF membrane was blocked with 5% skimmed milk for 2 h and further incubated with anti-UCHL1 primary antibody (1:2,000) for overnight at 4 °C. Subsequently, it was probed with anti-mouse secondary antibody (1:10,000) for 45 min. The membrane was developed using Luminata Forte reagent (Millipore, USA) and visualized under chemiluminescence system Image Quant (GE Healthcare, USA).
3
UCHL1 S-nitrosylation Chromatographic Analysis
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Recombinant UCHL1 (200 μl, 250 μM) was treated with different concentrations of GSNO for 2 h and loaded onto a Superdex 200, 10/30 (GE Healthcare Life Sciences, USA) gel filtration column. In case of cellular UCHL1 study, SH-SY5Y cells were treated with GSNO (500 nM) and rotenone (1 μM) for 16 h. Harvested cells were lysed in RIPA buffer (Sigma, USA) containing 1x Halt protease (Thermo Fisher Scientific, USA) and centrifuged at 16,000 × g for 20 min. The supernatant (200 μL, 1.5 mg/mL) was loaded onto a Superose 6 10/300 GL (GE Healthcare, USA) gel filtration column. Each chromatographic fraction was spotted on a nitrocellulose membrane using Bio-Rad dot blot apparatus. The membrane was incubated in 5% skimmed milk for 1 h. After blocking, membrane was incubated with anti-UCHL1 antibody (1:2,000) (Pierce, USA) for overnight. Finally, membrane was incubated with anti-mouse secondary antibody (1:10,000) for 45 min and was developed using Luminata Forte reagent (Millipore, USA) and immuno reactivity of protein was visualized under chemiluminescence system Image Quant (GE Healthcare Life Sciences, USA).
4
Western Blot Experimental Protocol
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2× Laemmli buffer was used to lyse the cells, followed by boiling at 95°C for 10 min. Boiled samples were resolved by SDS-PAGE followed by transfer onto polyvinylidenedifluoride or nitrocellulose membrane (Millipore). After transfer, the membrane was blocked with 5% skim milk followed by overnight incubation in primary antibody at 4°C. Following a 1-h wash after primary antibody incubation, HRP-conjugated secondary antibody was incubated for 1 h at room temperature, and the blot was washed before being developed for chemiluminescence signal using the Luminata Forte reagent (Millipore; WBLUF0500) and captured in the ImageQuant LAS-4000 gel documentation system (GE Healthcare). The dilutions used for the various primary and secondary antibodies were as follows: LIC1, 1:1,000; IC74, 1:1,000; HC, 1:500; Mad1, 1:500; Mad2, 1:250; p150Glued, 1:2,000; p50, 1:500; cyclin A, 1:200; cyclin B, 1:3,000, OD 157, 1:5,000, Zw10, 1:500; Hook2, 1:2,000, NudE1, 1:1,000; Nde1, 1:2,000; Lis1, 1:1,000; GST, 1:1,000; pin1, 1:1,000; β-actin, 1:2,000; myc, 1:2,000; BICD2, 1:2,500; LAMP1, 1:1,000; mCherry, 1:1,000; β-COP, 1:1,000; GM130, 1:1,000; anti-rabbit HRP, 1:10,000; and anti-mouse HRP, 1:10,000. The ImageJ software platform was used to perform all the densitometric analyses of immunoblot band intensities.
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