Slot blot apparatus

Manufactured by Bio-Rad

Sourced in United States

The Slot Blot Apparatus is a laboratory device used for transferring and immobilizing nucleic acid or protein samples onto a membrane for subsequent analysis. The apparatus features a vacuum-based system that allows for the simultaneous application of multiple samples onto the membrane, facilitating efficient and consistent sample transfer.

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Lab products found in correlation

Scion image, by Techcomp Instruments (4 mentions) Chemidoc, by Bio-Rad (3 mentions) Nitrocellulose membrane, by Bio-Rad (3 mentions) Hybond nylon membrane, by Cytiva (2 mentions) Ab27156, by Abcam (2 mentions) Chemidoc mp, by Bio-Rad (2 mentions) Protease inhibitor cocktail, by Roche (2 mentions) Anti myc antibody, by Thermo Fisher Scientific (2 mentions) Dnase rnase free water, by Thermo Fisher Scientific (2 mentions) Clarity ecl, by Bio-Rad (2 mentions) Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (2 mentions) Primary antibody anti dinitrophenylhydrazone primary or anti protein bound hne, by Merck Group (2 mentions) 51 mm polycarbonate centrifuge tubes, by Beckman Coulter (2 mentions) Sla 100.3 rotor, by Beckman Coulter (2 mentions) Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions) Pierce coomassie plus bradford protein assay, by Thermo Fisher Scientific (1 mentions) As132640, by Agrisera (1 mentions) Tp401, by AMS Biotechnology (1 mentions) Nitro blue tetrazolium chloride nbt, by Merck Group (1 mentions) 5 bromo 4 chloro 3 indolyl phosphate bcip, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Slot blot (12 citations) 48-well slot blot apparatus (3 citations)

35 protocols using slot blot apparatus

Actin Aggregates Profiling in Plants

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Whole seedlings, root base cells, and root tips containing stem cells were recollected and grinded in liquid N₂. Protein extracts were obtained with native lysis buffer (300 mM NaCl, 100 mM Hepes pH 7.4, 2 mM EDTA, 2% Triton X‐100) supplemented with 1X plant protease inhibitor (Sigma). Cellular debris was removed with 80,000 g centrifugation for 10 min at 4°C. Supernatant was recollected and protein concentration determined with Pierce Coomassie Plus (Bradford) Protein‐Assay (Thermo Scientific). A cellulose acetate membrane filter (GE Healthcare Life Sciences) was placed in a slot blot apparatus (Bio‐Rad) coupled to a vacuum system. Membrane was equilibrated with 3 washes with equilibration buffer (native buffer supplemented with 0.5% SDS). 200 µg of protein extract was supplemented with SDS at a final concentration of 0.5% and loaded and filter through the membrane. Then, the membrane was washed with 0.2% SDS. Retained aggregates of actin were detected using anti‐actin antibody [1:5,000] (Agrisera, AS132640). Extracts were also analyzed by SDS‐PAGE to determine total actin levels.

Llamas E., Torres‐Montilla S., Lee H.J., Barja M.V., Schlimgen E., Dunken N., Wagle P., Werr W., Zuccaro A., Rodríguez‐Concepción M, & Vilchez D. (2021). The intrinsic chaperone network of Arabidopsis stem cells confers protection against proteotoxic stress. Aging Cell, 20(8), e13446.

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Slot Blot Assay for Oxidative Biomarkers

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Protein carbonyls (PC) and protein-bound 4-hydroxy-2-trans-nonenal (HNE) were detected by slot blot assay [12 (link),13 (link)]. For determination of PC, samples were derivatized with 2,4-dinitrophenylhydrazine (DNPH) in advance. For determination of HNE and 3-NT, brain homogenates were prepared with Laemmli buffer. Protein (250 ng) from each sample were loaded onto a nitrocellulose membrane in separate wells in a slot-blot apparatus (Bio-Rad, Hercules, CA, USA) under vacuum formed by a water suction system. Membranes were blocked in 5% bovine serum albumin (BSA) in TBS-Tween20 (0.2% v/v) for 1.5 h and followed with incubation in primary antibody (anti-dinitrophenylhydrazone primary, anti-protein-bound HNE or anti 3-NT, respectively, each produced in rabbit, Sigma-Aldrich) for 2 h, washed three times in TBS-T and then incubated for 1 h with secondary antibody (goat anti-rabbit secondary linked to alkaline phosphatase). Membranes were developed for alkaline phosphatase activity (ALP) buffer containing 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) dipotassium and nitro blue tetrazolium (NBT) chloride, and then dried overnight, followed with image scanning for analysis performed using Scion Image (Scion Corporation, Frederick, MD).

Ren X., Hinchie A., Swomley A., Powell D.K, & Butterfield D.A. (2019). Profiles of Brain Oxidative Damage, Ventricular Alterations, and Neurochemical Metabolites in the Striatum of PINK1 Knockout Rats as Functions of Age and Gender: Relevance to Parkinson Disease. Free radical biology & medicine, 143, 146-152.

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Measurement of Protein Oxidation in Brain

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The slot-blot method was used to determine levels of protein carbonyls and protein-bound 4-hydroxynonenal (HNE) in brain. For protein carbonyl determination, samples were derivatized with 2,4-dinitrophenylhydrazine (DNPH). For HNE, samples were solubilized in Laemmli buffer. Protein (250 ng) from each sample was loaded onto a nitrocellulose membrane inrespective wells in a slot-blot apparatus (Bio-Rad) under vacuum. Membranes were blocked in 3% bovine serum albumin (BSA) in PBS with 0.2% (v/v) Tween-20 for 1.5 h and then incubatedin primary antibody (anti-dinitrophenylhydrazone primary or anti-protein-bound HNE, respectively, each produced in rabbit, Sigma-Aldrich) for 2 h, washed three times in PBS with 0.2% (v/v) Tween-20 and then incubated for 1 h with secondary antibody (goat anti-rabbit secondary linked to alkaline phosphatase). Membranes were developed with 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) dipotassium and nitro blue tetrazolium (NBT) chloride in alkaline phosphatase activity (ALP) buffer, dried, and scanned for analysis. Image analysis was performed using Scion Image (Scion Corporation, Frederick, MD).

Keeney J.T., Ren X., Warrier G., Noel T., Powell D.K., Brelsfoard J.M., Sultana R., Saatman K.E., Clair D.K, & Butterfield D.A. (2018). Doxorubicin-induced elevated oxidative stress and neurochemical alterations in brain and cognitive decline: protection by MESNA and insights into mechanisms of chemotherapy-induced cognitive impairment (“chemobrain”). Oncotarget, 9(54), 30324-30339.

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Polyglutamine Protein Aggregation Assay

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Human cell cultures were collected in non-denaturing lysis buffer (50 mm Hepes pH 7.4, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100) supplemented with EDTA-free protease inhibitor cocktail (Roche). Then, human cell samples were lysed by passing 10 times through a 27 G needle attached to a 1 ml syringe. Regarding C. elegans samples, the worms were collected with M9 buffer and worm pellets were frozen with liquid N₂. Frozen worm pellets were thawed on ice and worm extracts were generated by glass bead disruption. Then, either worm or cellular debris was removed with 8000g spin for 5 min and protein concentration was determined with BCA protein assay. Approximately 100 μg of protein extract was supplemented with SDS at a final concentration of 0.5% and loaded onto a cellulose acetate membrane assembled in a slot-blot apparatus (Bio-Rad). The membrane was then washed with 0.2% SDS. In human cells and C. elegans extracts, the retained polyQ-expanded proteins were assessed by immunoblotting for anti-polyQ-expansion diseases marker (Millipore, MAB1574, 1:5000) and anti-GFP antibodies (AMSBIO, #TP401, 1:5000), respectively. In addition, extracts were also analyzed by SDS–PAGE to determine the total levels of the corresponding proteins using the aforementioned antibodies.

Gutiérrez-Garcia R., Koyuncu S., Hommen F., Bilican S., Lee H.J., Fatima A, & Vilchez D. (2023). G3BP1-dependent mechanism suppressing protein aggregation in Huntington’s models and its demise upon stress granule assembly. Human Molecular Genetics, 32(10), 1607-1621.

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Quantification of RNA-DNA Hybrids by Slot Blot

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Hybond Nylon membrane (Amersham) was pre-soaked in TE and a slot blot apparatus was assembled according to manufacturer’s instructions (Bio-Rad). Samples with matching RNase H1-digested controls were added to the blot in decreasing amounts, and nucleic acids were crosslinked to the membrane with a Strategene UV Stratalinker 1800 using the auto crosslink setting. Blots were blocked in milk, incubated with S9.6 (1:2,000) followed by mouse-HRP and imaged in a Bio-Rad Chemidoc MP. After imaging the R-loops, blots were stripped and re-probed using a dsDNA-specific antibody (Abcam ab27156) at 1:20,000. Intensities were measured with ImageJ,^{62 (link)} and normalized intensity was obtained by dividing the S9.6 signal by the dsDNA signal.^{63 (link)} A standard plot was made for each sample and antibody, and samples were chosen for analysis when their intensity was linear.

Munden A., Benton M.L., Capra J.A, & Nordman J.T. (2022). R-loop Mapping and Characterization During Drosophila Embryogenesis Reveals Developmental Plasticity in R-loop Signatures. Journal of molecular biology, 434(13), 167645.

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SNO Quantification in AD Mice Brains

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Brain tissues from WT (N = 4) and AD (N = 4) mice were homogenized with the presence of NEM and incubated with 5% SDS for 0.5 h at room temperature. BCA assay was used to determine protein concentrations. An aliquot of 250 μL PBS containing 0.5 μg protein was loaded onto a nitrocellulose membrane with a slot blot apparatus (Bio-Rad). The membranes were blocked with 3% (w/v) BSA solution and 5 mM NEM overnight at 4°C and incubated with a 1:2500 dilution of anti-SNO antibody produced by mouse (Sigma) for 2 hours. After rinsing the membrane, anti-mouse IgG alkaline phosphatase secondary antibody (Sigma) was added with the dilution factor 1:5000 and incubated with the membrane for 1 hour. The membrane was washed in wash blot (PBS with 0.1% Tween 20) and developed using 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Thermo Fisher)/nitro blue tetrazolium chloride (NBT) (Sigma) colorimetric development. The blot was dried, scanned and slot profiles quantified using Scion Image. Statistical testing (student’s t-test) was performed in Origin 8.0. The entire experiment was repeated twice.

Gu L, & Robinson R.A. (2016). High-throughput Endogenous Measurement of S-Nitrosylation in Alzheimer’s Disease using Oxidized Cysteine-selective cPILOT. The Analyst, 141(12), 3904-3915.

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Quantifying Tau Protein Aggregation by Slot Blot

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Six μM of p-tau was incubated in the standard aggregation buffer (see above) without ThS for 0, 48 or 120 hours. Two μL of the samples were diluted into 400 μL TBS buffer (20 mM Tris, pH 7.4, 150 mM NaCl) and applied to a PVDF membrane in a slot blot apparatus (Bio-Rad, Hercules, CA). The membrane was blocked in 5% nonfat milk in TBST (20 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween 20) for 1 hour, and then incubated with the MC-1 monoclonal antibody (at 1:1000 dilution) or MAB3494 (at 1:3000 dilution) for overnight at 4°C. After washing three times in TBST, the membrane was incubated with HRP goat anti-mouse secondary antibody (at 1:10000 dilution) for 1 hour at room temperature. The membrane was washed again 3 times in TBST and developed with the Lumi-light Western Blotting Substrate for 5 min.

Liu M., Sui D., Dexheimer T., Hovde S., Deng X., Wang K.W., Lin H.L., Chien H.T., Kweon H.K., Kuo N.S., Ayoub C.A., Jimenez-Harrison D., Andrews P.C., Kwok R., Bochar D.A., Kuret J., Fortin J., Tsay Y.G, & Kuo M.H. (2020). Hyperphosphorylation Renders Tau Prone to Aggregate and to Cause Cell Death. Molecular neurobiology, 57(11), 4704-4719.

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Quantitative DNA Methylation Analysis

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Serially diluted genomic DNA was denatured and loaded onto nitrocellulose membrane using a slot blot apparatus (Biorad). After washing with 4X saline sodium citrate, the membrane was subjected to UV crosslinking and probed with a 5-methylcytosine antibody (C15200081, Diagenode). After chemiluminescence detection, the membrane was stained with 0.2% methylene blue in 0.3M sodium acetate (pH 5.2) as a loading control for the slot blot.

Kan G., He H., Zhao Q., Li X., Li M., Yang H, & Kim J.K. (2017). Functional dissection of the role of UHRF1 in the regulation of retinoblastoma methylome. Oncotarget, 8(24), 39497-39511.

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Secreted Sema3F and Sema3A Detection

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To detect secreted ^MycSema3F protein in cultured medium, 14 DIV primary cortical cultures derived from E14.5 ^Myc/MycSema3F embryos were treated with TTX or bicuculline for 48 hrs. Culture supernatants were collected and subjected to rabbit-anti-Myc immunoprecipitation and analyzed by Western blot with a mouse antibody directed against Myc. To detect secreted Sema3F from wild type cortical cultures, 14 DIV wild type primary cortical neurons were treated with TTX or bicuculline for 48 hrs. Supernatants were centrifuged, collected, filtered and loaded into a slot-blot apparatus (Bio-Rad). The resulting membranes were incubated with AP-Npn-2^Ecto (5 nM), for 1 hour at room temperature. NBT/BCIP was used as a substrate to detect the presence of the alkaline phosphatase signal. An analogous approach was used to detect Sema3A.

Wang Q., Chiu S.L., Koropouli E., Hong I., Mitchell S., Easwaran T.P., Hamilton N.R., Gustina A.S., Zhu Q., Ginty D.D., Huganir R.L, & Kolodkin A.L. (2017). Neuropilin-2/PlexinA3 Receptors Associate with GluA1 and Mediate Sema3F-dependent Homeostatic Scaling in Cortical Neurons. Neuron, 96(5), 1084-1098.e7.

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Phosphorylated Peptide Detection via Filter-Trap

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We modified the filter-trap analysis as described previously [26 (link)]. Briefly, phosphorylated or non-phosphorylated peptides were diluted with TBS (Tris–HCl, pH 7.5, 150 mM NaCl), and applied to a 0.22-μm cellulose acetate membrane (Millipore) on a slot blot apparatus (Bio-Rad, Hercules, CA, USA) using a vacuum manifold. After washing with TBS containing 0.1% Triton X-100, the membrane was incubated with anti-phosphorylated p62 and pan-p62 antibodies and detected using the ECL detection system as described above.

Tanji K., Miki Y., Ozaki T., Maruyama A., Yoshida H., Mimura J., Matsumiya T., Mori F., Imaizumi T., Itoh K., Kakita A., Takahashi H, & Wakabayashi K. (2014). Phosphorylation of serine 349 of p62 in Alzheimer’s disease brain. Acta Neuropathologica Communications, 2, 50.

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