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Vacuum slot blot manifold

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The Vacuum slot-blot manifold is a laboratory equipment designed for the efficient transfer of biomolecules, such as proteins or nucleic acids, onto membranes. The core function of this device is to create a vacuum-assisted system that allows for the simultaneous application of samples across multiple wells or slots, enabling rapid and uniform sample transfer.

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Lab products found in correlation

Chemidoc xrs plus analyzer, by Bio-Rad (3 mentions) Nvt 90 rotor, by Beckman Coulter (2 mentions) Mouse anti dsdna, by Abcam (1 mentions) Anti ubiquitin, by Abcam (1 mentions) Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions) Anti ha antibody, by Cell Signaling Technology (1 mentions) Benzonase nuclease, by Merck Group (1 mentions) Anti parp1, by BD (1 mentions) Nitrocellulose membrane, by Bio-Rad (1 mentions) Nitrocellulose, by Bio-Rad (1 mentions) Nylon membrane, by GE Healthcare (1 mentions) Flamingo fluorescent protein gel stain, by Bio-Rad (1 mentions) Benzonase, by Merck Group (1 mentions)

Close spelling variants of this product

Slot blot (12 citations) Slot blot manifold (3 citations)

7 protocols using vacuum slot blot manifold

1

DNA Blotting and Protein Detection

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DNA was detected by blotting the DNA with a slot blot vacuum manifold (Biorad) onto a positively charged nylon membrane. The membrane was probed with mouse anti-dsDNA (Abcam) (1:2000). Specific proteins were detected by normalizing to DNA concentration and digesting with benzonase. The proteins were separated on a polyacrylamide gel and silver stained (Sigma).
Bhargava V., Goldstein C.D., Russell L., Xu L., Ahmed M., Li W., Casey A., Servage K., Kollipara R., Picciarelli Z., Kittler R., Yatsenko A., Carmell M., Orth K., Amatruda J.F., Yanowitz J.L, & Buszczak M. (2019). GCNA preserves genome integrity and fertility across species. Developmental cell, 52(1), 38-52.e10.
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2

Top1-DNA Covalent Complex Isolation

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Top1-DNA covalent complexes were isolated using the immunocomplex of enzyme (ICE) bioassay as previously described 28 . Briefly, cells or tissues were lysed in 1% sarkosyl with dounce homogenization (10 strokes for cells, 40 strokes for embryonic CNS tissue). DNA was sheered through a 26G needle (10 strokes) and cell lysates were gently layered onto a CsCl cushion and centrifuged in a NVT 90 rotor at 122 000 × g for 20 h at 25°C (Beckman-Coulter). The resulting pelleted covalent DNA-protein complexes were washed, resuspended in TE, and aliquots were diluted with 25 mM sodium phosphate buffer (pH 6.5) and applied to a nitrocellulose membrane by using a slot-blot vacuum manifold (Bio-Rad). Total protein extract was applied to adjacent slots as a control for total Top1 levels amongst tissues. Top1 protein and Top1-DNA complexes were immunodetected using an anti-Top1 polyclonal antibody (rabbit, 1:1000, Bethyl, cat# 302-590A), followed by HRPconjugated anti-rabbit secondary antibody and detection using ECL Prime on X-ray film. ICE blots were subsequently probed with 32P-labeled mouse (ES) or human (293T) genomic DNA (gDNA) to control for relative DNA loading. For astrocyte ICE assays, densitometric analysis of the Top1-DNA signal was performed using Image J. Experiments were performed at least in duplicate.
Katyal S., Lee Y., Nitiss K.C., Downing S.M., Li Y., Shimada M., Zhao J., Russell H.R., Petrini J.H., Nitiss J.L, & McKinnon P.J. (2014). Aberrant Topoisomerase-1-DNA Lesions are Pathogenic in Neurodegenerative Genome Instability Syndromes. Nature neuroscience, 17(6), 813-821.
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3

Top1-DNA Covalent Complex Isolation

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Top1-DNA covalent complexes were isolated using the immunocomplex of enzyme (ICE) bioassay as previously described 28 . Briefly, cells or tissues were lysed in 1% sarkosyl with dounce homogenization (10 strokes for cells, 40 strokes for embryonic CNS tissue). DNA was sheered through a 26G needle (10 strokes) and cell lysates were gently layered onto a CsCl cushion and centrifuged in a NVT 90 rotor at 122 000 × g for 20 h at 25°C (Beckman-Coulter). The resulting pelleted covalent DNA-protein complexes were washed, resuspended in TE, and aliquots were diluted with 25 mM sodium phosphate buffer (pH 6.5) and applied to a nitrocellulose membrane by using a slot-blot vacuum manifold (Bio-Rad). Total protein extract was applied to adjacent slots as a control for total Top1 levels amongst tissues. Top1 protein and Top1-DNA complexes were immunodetected using an anti-Top1 polyclonal antibody (rabbit, 1:1000, Bethyl, cat# 302-590A), followed by HRPconjugated anti-rabbit secondary antibody and detection using ECL Prime on X-ray film. ICE blots were subsequently probed with 32P-labeled mouse (ES) or human (293T) genomic DNA (gDNA) to control for relative DNA loading. For astrocyte ICE assays, densitometric analysis of the Top1-DNA signal was performed using Image J. Experiments were performed at least in duplicate.
Katyal S., Lee Y., Nitiss K.C., Downing S.M., Li Y., Shimada M., Zhao J., Russell H.R., Petrini J.H., Nitiss J.L, & McKinnon P.J. (2014). Aberrant Topoisomerase-1-DNA Lesions are Pathogenic in Neurodegenerative Genome Instability Syndromes. Nature neuroscience, 17(6), 813-821.
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4

Detect DNA-Protein Crosslinks by Slot Blot

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Specific DPCs were detected using a vacuum slot-blot manifold (Bio-Rad) followed by immunodetection. DPCs were visualized using the Bio-Rad ChemiDoc XRS Plus Analyzer. See the Supplemental Experimental Procedures for details.
Vaz B., Popovic M., Newman J.A., Fielden J., Aitkenhead H., Halder S., Singh A.N., Vendrell I., Fischer R., Torrecilla I., Drobnitzky N., Freire R., Amor D.J., Lockhart P.J., Kessler B.M., McKenna G.W., Gileadi O, & Ramadan K. (2016). Metalloprotease SPRTN/DVC1 Orchestrates Replication-Coupled DNA-Protein Crosslink Repair. Molecular Cell, 64(4), 704-719.
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5

Slot-Blot Immunoblotting Protocol for DNA Detection

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For immunoblotting, samples of genomic DNA were diluted in 200 μl 1X TBE and applied to a nitrocellulose membrane (Bio-Rad) using a vacuum slot-blot manifold (Bio-Rad). To reduce sample viscosity and to avoid membrane clogging, aliquots of genomic DNA were treated with 20 units of benzonase nuclease (Sigma-Aldrich) for 30 min at 37 °C prior to loading onto nitrocellulose membrane. The membrane was then blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBS-T) and then probed either with anti-PARP-1 (BD Pharmingen), anti-HA antibody (Cell Signaling Technology) or with anti-ubiquitin (Abcam) antibody, as indicated in the figure legends. Goat anti-mouse or goat anti-rabbit IgG conjugated to horseradish peroxidase (Bio-Rad) was used as secondary antibody, and immobilized horseradish peroxidase activity was detected by enhanced chemiluminescence (Thermo Fisher Scientific).
Prasad R., Horton J.K., Dai D.P, & Wilson S.H. (2018). Repair pathway for PARP-1 DNA-protein crosslinks. DNA repair, 73, 71-77.
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6

Slot-Blot Protein Quantification Protocol

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Isolates, typically containing 200–500 ng DNA, were diluted in 25 mM sodium phosphate (pH 6.5) or TBS (Tris-Buffered Saline: 10 mM Tris, pH 7.5, 150 mM NaCl) and applied to nitrocellulose (Bio-Rad) or PDVF (Millipore) membranes using a vacuum slot-blot manifold (Bio-Rad). Membranes were blocked in 0.5% alkali-soluble casein (Novagen) dissolved in TBST (TBS containing 0.1% Tween-20) for at least 1 h; incubated with primary antibodies at least 3 h at room temperature or overnight at 4°C; and with secondary HRP-conjugated antibodies for 1 h at room temperature. After each incubation, membranes were washed three times for 5 min with TBST. Antibody signal was quantified by imaging the membrane on a Bio-Rad ChemiDoc XRS Plus Analyzer.
Kiianitsa K, & Maizels N. (2014). Ultrasensitive isolation, identification and quantification of DNA–protein adducts by ELISA-based RADAR assay. Nucleic Acids Research, 42(13), e108.
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7

Quantification and Detection of DNA-Protein Crosslinks

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Total DPCs were visualized by Flamingo™ Fluorescent Protein Gel Stain (Bio-Rad) as recommended by the manufacturer after electrophoretic separation on polyacrylamide gels. Specific DPCs were detected by immuno-staining. In brief, dsDNA containing the DPCs was quantified using a Quant-iT™ PicoGreen™ dsDNA assay kit. Normalized amounts of DNA were digested with benzonase (Merck Millipore) for 1 hour at 37°C. Samples were diluted in Tris-buffered saline (TBS) and applied to a PVDF membrane using a vacuum slot-blot manifold (Bio-Rad). The membrane was then blocked in 3% BSA in TBS/T (TBS, 0.1% Tween-20) and incubated with primary antibodies followed by incubation with HRPconjugated secondary antibodies. DPCs were visualized using a Bio-Rad ChemiDoc XRS Plus Analyzer. For slot-blot detection of dsDNA, 10-20 μg of DNA were incubated with proteinase K to digest the crosslinked proteins, diluted in Tris/Borate/EDTA (TBE) buffer and applied to nylon membrane (GE Healthcare). The membrane was blotted with an anti-dsDNA antibody and developed as before.
Vaz B., Ruggiano A., Popović M., Rodríguez‐Berriguete G., Kilgas S., Singh A.N., Higgins G.S., Kiltie A.E., & Ramadan K. (2020). SPRTN protease and SUMOylation coordinate DNA-protein crosslink repair to prevent genome instability.
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