Kwikquant western blot detection kit

Manufactured by Kindle Biosciences

The KwikQuant Western Blot Detection Kit is a laboratory product designed to facilitate the detection and quantification of proteins using the Western blot technique. The kit provides the necessary reagents and materials to perform this analytical procedure.

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Lab products found in correlation

Kwikquant imager, by Kindle Biosciences (7 mentions) Phenyl methyl sulfonyl fluoride (pmsf), by Cell Signaling Technology (3 mentions) Ripa buffer, by Cell Signaling Technology (3 mentions) Benzonase, by Merck Group (3 mentions) Protease inhibitor cocktail, by Roche (3 mentions) Trans blot turbo transfer system, by Bio-Rad (2 mentions) Anti α tubulin, by Developmental Studies Hybridoma Bank (2 mentions) Anti flag m2, by Merck Group (2 mentions) Hrp conjugated goat anti rabbit secondary antibody, by Kindle Biosciences (2 mentions) Nupage mes sds running buffer, by Thermo Fisher Scientific (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions) Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions) Hyglo development solution, by Thomas Scientific (1 mentions) Anti h3, by Abcam (1 mentions) Anti flag, by Merck Group (1 mentions) Bradford assay, by Bio-Rad (1 mentions)

9 protocols using kwikquant western blot detection kit

SirT1 Protein Immunoprecipitation and Western Blot

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Proteins were separated on NuPAGE 4–12% Bis-Tris protein gels
using NuPAGE MES SDS running buffer (Thermo Fisher, Waltham, MA) and transferred
on PVDF membranes using Trans-Blot Turbo transfer system (Bio-Rad Laboratories,
Hercules, CA). Membranes were blocked with 5% nonfat milk, incubated with
primary antibodies overnight, washed three times with TBST, and probed with
respective secondary antibodies for 1 hour at room temperature.
For immunoprecipitation, cell lysates were rotated at 4 °C
overnight with 2 μg of SirT1 antibody that had been pre-coupled with 20
μl of protein A/G beads for 1 hour. Beads were rinsed and proteins
separated by SDS-polyacrylamide gel electrophoresis. Overexpressed FLAG-SirT1
was directly immunoprecipitated with anti-FLAG antibody coated magnetic beads.
Protein-protein interactions were identified by blotting for the respective
interacting protein using GAPDH, SirT1, and Ac-p53 antibodies. Membranes were
developed using the KwikQuant Western blot detection kit and images were
recorded using the KwikQuant imager (Kindle Biosciences, Greenwich, CT).

Rizvi S.H., Shao D., Tsukahara Y., Pimentel D.R., Weisbrod R.M., Hamburg N.M., McComb M.E., Matsui R, & Bachschmid M.M. (2021). Oxidized GAPDH transfers S-glutathionylation to a nuclear protein Sirtuin-1 leading to apoptosis. Free radical biology & medicine, 174, 73-83.

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Quantifying 3xFLAG Protein in Ovary

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To assay 3xFLAG MH protein abundance in the ovary, we dissected 20 ovary pairs in 1X PBS and ground the material in RIPA buffer (Cell Signaling Technology, Danvers, MA), Protease Inhibitor Cocktail (Roche, Basel, Switzerland), and 2X PMSF (Cell Signaling Technology, Danvers, MA). To promote solubility, we incubated the lysate in benzonase (Sigma Aldrich, St. Louis, MO) for 1hr at 4C. We used 20μg of lysate and probed with 1:10,000 anti-FLAG (M2, Sigma Aldrich, St. Louis, MO) or 1:1000 anti-αTubulin (Developmental Studies Hybridoma Bank, Iowa City, IA) and 1:1000 anti-mouse HRP secondary antibodies (Kindle Biosciences, Greenwich, CT). We exposed blots with Kwikquant Western Blot detection kit and imaged with a Kwikquant imager (Kindle Biosciences, Greenwich, CT).

Brand C.L, & Levine M.T. (2022). Cross-species incompatibility between a DNA satellite and the Drosophila Spartan homolog poisons germline genome integrity. Current biology : CB, 32(13), 2962-2971.e4.

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Western Blot Analysis of Cell Cycle Proteins

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A total of 100 oocytes were mixed with SDS sample buffer (1% SDS, 1% β‐mercaptoethanol, 20% glycerol, 50 mM Tris‐HCl (pH 6.8) and the phosphatase inhibitors sodium fluoride (25 mM) and sodium orthovanadate (1 mM) and denatured at 95℃ for 5 minutes. Proteins were separated by electrophoresis in 10% SDS polyacrylamide precast gel (Bio‐Rad, #4561036). The separated proteins were transferred to nitrocellulose membranes (Bio‐Rad, #1704156) using a Trans‐Blot Turbo Transfer System (Bio‐Rad) and then blocked with 2% ECL blocking (Amersham, #RPN418) solution in TBS‐T (Tris‐buffered saline with 0.1% Tween 20) for 1h. The membranes were incubated overnight at 4°C with primary antibody to detect MAD2 (1:500) and ZW10 (1:500), Securin (1:500) or for 1 h to detect α‐Tubulin (1:500). The membranes were incubated with secondary antibody (1:1000; Kindle Bioscience #R1006) for 1 h at room temperature. The signals were detected using ECL Select western blotting detection reagents (Kindle Biosciences, KwikQuant Western Blot Detection Kit) following the manufacturer's protocol. Images were analyzed using Image J software (NIH) (Schneider et al., 2012 (link)) and were normalized to α‐Tubulin and set to 1 in WT.

Blengini C.S., Nguyen A.L., Aboelenain M, & Schindler K. (2021). Age‐dependent integrity of the meiotic spindle assembly checkpoint in females requires Aurora kinase B. Aging Cell, 20(11), e13489.

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Western Blot Analysis of Yeast Proteins

Cited 2 times

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The assay was performed as previously described (Hildebrandt, Cheng et al. 2016 (link); Berger, Kim et al. 2018 (link); Schey, Buttery et al. 2021 (link)). Whole cell lysates of mid log cells were prepared and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE; 6% stacking with 9.5% resolving gel) then transferred onto nitrocellulose. Blots were blocked with 5% milk then sequentially incubated with rabbit anti-Ydj1 primary antibody (courtesy of A. Caplan) and HRP-conjugated goat antirabbit secondary antibody (Kindle Biosciences, Greenwich, CT). Fluorescence was detected using the KwikQuant Western Blot Detection Kit (Kindle Biosciences) and a KwikQuant Imager and as per manufacturer's instructions. Protein bands were quantified using NIH ImageJ, and resulting values were used for calculating ratios for prenylated and nonprenylated bands. The blots containing yeast Nap1 were treated similarly but incubated with mouse anti-His primary antibody (Thermo Fisher Scientific, Waltham, MA) and HRP-conjugated sheep antimouse secondary antibody (Kindle Biosciences, Greenwich, CT).

Kim J.H., Hildebrandt E.R., Sarkar A., Yeung W., Waldon L.R., Kannan N, & Schmidt W.K. (2023). A comprehensive in vivo screen of yeast farnesyltransferase activity reveals broad reactivity across a majority of CXXX sequences. G3: Genes|Genomes|Genetics, 13(7), jkad094.

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Immunoprecipitation and Western Blot Analysis

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Total lysates were prepared as described above. For immunoprecipitations, equal amounts of proteins were pre-cleared with 15–20 μl TrueBlot anti-Rabbit or anti-Mouse Ig IP agarose beads (Rockland) and incubated with the indicated antibodies for 1 h at RT. After incubation, 30 μl of TrueBlot agarose beads was added to each immune reaction and rotated at 4 °C overnight. The immunopellets were then washed 3 times with 1X RIPA buffer and boiled in 40 μl loading buffer. Proteins were separated on NuPage 4–12% Bis Tris gels (Thermo Fisher scientific), transferred to nitrocellulose membranes, and analyzed by immunoblotting using specific primary antibodies, followed by secondary anti-IgG antibodies conjugated with HRP (GE Healthcare). For immunoprecipitations, mouse or rabbit Trueblot Ig HRP secondary antibodies (Rockland) were used. Immunoreactive bands were detected by chemiluminescence using KwikQuant Western Blot Detection kit and Kwikquant Imager acquisition system (Kindle Biosciences).

Sanchez-Solana B., Wang D., Qian X., Velayoudame P., Simanshu D.K., Acharya J.K, & Lowy D.R. (2021). The tumor suppressor activity of DLC1 requires the interaction of its START domain with Phosphatidylserine, PLCD1, and Caveolin-1. Molecular Cancer, 20, 141.

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Quantitative Immunoblotting of Yeast Proteins

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Yeast were cultured to log phase (A₆₀₀ 0.75–1.0) in selective SC-uracil at 25° unless otherwise noted. Cell pellets of equal mass were harvested by centrifugation, washed with water, and processed by alkaline hydrolysis and trichloroacetic acid precipitation (Kim et al. 2005 (link)). Total cell precipitates were resuspended in urea-containing Sample Buffer (250 mM Tris, 6 M urea, 5% β-mercaptoethanol, 4% SDS, 0.01% bromophenol blue; pH 8), and analyzed by SDS-PAGE and immunoblotting. Blots were processed according to standard protocols using appropriate dilutions of rabbit anti-Ydj1p (courtesy of Dr. Avrom Caplan) and HRP-conjugated donkey or goat anti-rabbit antibodies (GE Healthcare, Little Chalfont, UK; Kindle Biosciences, Greenwich, CT). Antibody dilutions were prepared using TBST (10 mM Tris, 150 mM NaCl, 0.1% Tween-20; pH 7.5) containing 1% milk (w/v). Immune complexes on blots were detected using X-ray film after treatment with HyGLO development solution (Denville Scientific, South Plainfield, NJ) or using a KwikQuant Imager at multiple exposure times after treatment with the KwikQuant Western Blot Detection Kit (Kindle Biosciences).

Berger B.M., Kim J.H., Hildebrandt E.R., Davis I.C., Morgan M.C., Hougland J.L, & Schmidt W.K. (2018). Protein Isoprenylation in Yeast Targets COOH-Terminal Sequences Not Adhering to the CaaX Consensus. Genetics, 210(4), 1301-1316.

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Quantitative Western Blot Analysis of Yeast Ydj1p

Cited 1 time

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Whole cell lysates of mid log yeast were prepared as previously
described, separated by sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS–PAGE) (14%), and transferred onto nitrocellulose,
and blots were incubated with the rabbit anti-Ydj1p primary antibody (courtesy
of A. Caplan) and HRP-conjugated goat anti-rabbit secondary antibody (Kindle
Biosciences, Greenwich, CT).^{18 (link),52 (link)} Immune complexes were detected
using a KwikQuant Imager at multiple exposure times after development of blots
with the KwikQuant Western Blot Detection Kit (Kindle Biosciences).

Ashok S., Hildebrandt E.R., Ruiz C.S., Hardgrove D.S., Coreno D.W., Schmidt W.K, & Hougland J.L. (2020). Protein Farnesyltransferase Catalyzes Unanticipated Farnesylation and Geranylgeranylation of Shortened Target Sequences. Biochemistry, 59(11), 1149-1162.

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Quantifying Ovarian Protein Levels

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To assay protein abundance in the ovary, we dissected 20 ovary pairs in 1XPBS and ground the material in 100 µL of RIPA buffer (Cell signaling technology, Danvers, MA), 0.4 µL protease inhibitor cocktail (Roche, Basel, CH), and 1 µL of 2x PMSF (Cell signaling technology, Danvers, MA). To promote solubility of this heterochromatin-bound protein, we incubated the lysate in 0.5 µL of Benzonase (Sigma Aldrich, St. Louis, MO) for 1 hr at 4C. After centrifuging briefly to remove debris, we quantified using a Bradford assay (Bio-Rad, Hercules, CA) and ran 20 µg of lysate in each lane. We probed with anti-Flag (Sigma Aldrich, St. Louis, MO) at 1:10,000 or anti-H3 (Abcam, Cambridge, UK) at 1:5000 and anti-mouse or anti-rabbit HRP secondaries (Kindle Biosciences, Greenwich, CT, both 1:1000). We exposed blots with Kwikquant Western Blot detection kit (Kindle Biosciences, Greenwich, CT) and imaged with a Kwikquant imager (Kindle Biosciences, Greenwich, CT).

Saint-Leandre B., Christopher C, & Levine M.T. (2020). Adaptive evolution of an essential telomere protein restricts telomeric retrotransposons. eLife, 9, e60987.

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Quantifying 3x-FLAG MH Protein in Ovary

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To assay 3x-FLAG MH protein abundance in the ovary, we dissected 20 ovary pairs in 1X PBS and ground the material in RIPA buffer (Cell Signaling Technology, Danvers, MA), Protease
Inhibitor Cocktail (Roche, Basel, Switzerland), and 2X PMSF (Cell Signaling Technology, Danvers, MA). To promote solubility, we incubated the lysate in benzonase (Sigma Aldrich, St.
Louis, MO) for 1hr at 4C. We used 20µg of lysate and probed with 1:10,000 anti-FLAG (M2, Sigma Aldrich, St. Louis, MO) or 1:1000 anti-αTubulin (Developmental Studies Hybridoma Bank, Iowa City, IA) and 1:1000 anti-mouse HRP secondaries (Kindle Biosciences, Greenwich, CT).
We exposed blots with Kwikquant Western Blot detection kit and imaged with a Kwikquant imager (Kindle Biosciences, Greenwich, CT).

Brand C.L., & Levine M.T. (2021). Cross-species incompatibility between a DNA satellite and the Drosophila Spartan homolog poisons germline genome integrity.

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