WT and FAIM KO HeLa cells were transiently transfected with an eGFP-tagged native human SOD1 or aggregation-prone human SOD1-G93A protein expression vector, and fluorescently tagged cells were then harvested at 48 h. Cells were washed with PBS and then lysed in PBS containing 2% SDS, 1 mM MgCl2, protease inhibitor cocktail and 25 unit/ml Benzonase (Merck). Protein concentrations were quantified using 660 nm Protein Assay Reagent with Ionic Detergent Compatibility Reagent (IDCR) (Thermo Scientific). Equal amounts of protein extracts underwent vacuum filtration through a 0.2 μm pore size cellulose acetate membrane (GE Healthcare) for the detection of SOD1 aggregates using a 96 well format Dot-Blot apparatus (Bio-Rad). The membrane was washed twice with 0.1% SDS in PBS and western blotted using anti-GFP antibody (Cell Signaling Technology) to detect aggregated proteins. Cell free aggregates of SOD1-G93A were similarly applied to nitrocellulose membrane. In cell-free experiments, SOD1 protein was vacuum filtered as above, after which membranes were western blotted using anti-SOD1 antibody (Cell Signaling Technology) to detect aggregated proteins.
Dot blot apparatus
Manufactured by Bio-Rad
Sourced in United States, Germany
The Dot Blot Apparatus is a laboratory tool used for the analysis of biomolecules, such as proteins or nucleic acids. Its core function is to efficiently immobilize and transfer these biomolecules onto a membrane or filter, enabling further downstream analysis and detection techniques.
Automatically generated - may contain errors
Lab products found in correlation
Hybond, by Cytiva (16 mentions)
Whatman, by Cytiva (6 mentions)
Imagequant software, by GE Healthcare (4 mentions)
Typhoon trio imager, by GE Healthcare (4 mentions)
Nitrocellulose membrane, by Merck Group (3 mentions)
Protran, by Cytiva (3 mentions)
Methylene blue, by Merck Group (3 mentions)
Hybond xl membrane, by Cytiva (3 mentions)
Typhoon biomolecular imager, by GE Healthcare (3 mentions)
Nitrocellulose membrane, by Cytiva (3 mentions)
Rnase inhibitor, by Cytiva (3 mentions)
Benzonase, by Merck Group (2 mentions)
Ionic detergent compatibility reagent, by Thermo Fisher Scientific (2 mentions)
Las 1000 image analyzer, by Fujifilm (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Luminata forte western hrp substrate, by Merck Group (2 mentions)
Saline sodium citrate solution, by Merck Group (2 mentions)
N membrane, by GE Healthcare (2 mentions)
S9.6 antibody, by Abcam (2 mentions)
Imagequant, by GE Healthcare (2 mentions)
85 protocols using dot blot apparatus
1
Detecting SOD1 Aggregates in Cells
2
Quantitative Fluorescent Maleimide Assay
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This assay was modified from the previous reports [34 (link)]. Proteins treated with or without the indicated stimuli were allowed to react with Alexa Fluor 680C2 maleimide (red fluorescence at the final concentration of 10 μM) at 4 °C for 2 h. After the interaction, the protein samples were either directly applied to the PVDF membrane in a Bio-Rad dot-blot apparatus or subjected to SDS-PAGE separation. The signal of fluorescent maleimide in the membranes was captured with a Fujifilm image LAS-1000 analyzer (Fujifilm, Tokyo, Japan) and quantified with ImageJ software. EZ blue staining was performed to confirm the equal loading of proteins.
3
Dot Blot and Western Blot Assay
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Bacterial suspension (20 µg protein/strain/well), MAA-BSA, BSA, MAA-LDL, AaHSP60 and Rgp44 (5 µg/well) in 200 µl of TBS, pH7.6, were loaded onto TBS pre-wetted nitrocellulose membrane using dot blot apparatus (Bio-Rad). MAA-BSA, MDA-BSA and PC-BSA (10 µg protein/lane) or Pg bacterial proteins (25 µg protein/strain/lane) were separated by SDS-PAGE and blotted onto nitrocellulose membranes for Western blotting. All blots were blocked in 5% BSA-TBS, incubated with Ab in 5% BSA-0.05% Tween 20-TBS and washed with 0.05% Tween 20-TBS. The binding to 4D5-D5 and 4F11-E2, 2.5 µg/ml in Dot blot and 0.5 µg/ml in Western blot was visualised by using goat-anti-mouse IgG IRDye800 (0.25 µg/ml; LI-COR Biosciences, Lincoln, NE) as secondary Ab. Isotype control Ab (cIgG, 2.5 µg/ml) was used as a reference. The fluorescent signals were detected with an Odyssey IR imager and Image Studio™ Software (LI-COR Biosciences).
4
Immunoblot Detection of Proteins
5
Quantitative Tau Protein Detection
6
Assessing rAAV9-GFP Capsid Stability
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rAAV9-GFP was diluted to 2 ng/µl in citrate-phosphate buffer at pH 7.4, 6.0, 5.5, or 4.0 containing 150 mmol/l NaCl. Samples were incubated in a BioRad (Hercules, CA) C1000 Touch Thermal Cycler at a temperature range of 4–100 °C for initial experiments to broadly determine capsid stability (data not shown) then a narrower temperature range of 70–80 °C for subsequent experiments to more accurately assess capsid stability, for 5 minutes, then cooled to 4 °C. Twenty ng of a treated sample was immobilized onto a nitrocellulose membrane using a dot-blot apparatus (Bio-Rad). Blots were blocked in 5% milk in 0.05% Tween-PBS and probed for intact capsids, denatured capsids, or VP1u externalization using anti-AAV9 capsid monoclonal antibodies HL-2370 at 1:1,000, B1 (American Research Products, Waltham, MA) at 1:3,000, or A1 (American Research Products) at 1:20, respectively. Detection was carried out using HRP-linked anti-mouse monoclonal antibody at 1:5,000 (GE Healthcare) and Immobilon chemiluminescent substrate (Millipore, Billerica, MA)
7
Maleimide-based Thiol Quantification Assay
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A maleimide-labeling assay for -SH groups was performed as previously reported [25 (link),26 (link),27 (link)]. Proteins from feces, serum and organs (kidney, bladder and colon) were allowed to react with 5 μM Alexa Fluor 680 C2 maleimide at 4 °C for 2 h. After the reaction, the protein samples were either subjected to SDS-PAGE separation or directly applied to the PVDF membrane in a Bio-Rad dot-blot apparatus. The fluorescent signals of the labeled maleimide in the gel or membranes were captured with a Fujifilm image LAS-4000 analyzer (Fujifilm, Tokyo, Japan) and quantified with ImageJ software. The equal loading of proteins in each lane was verified by EZ blue staining.
8
Analyzing Protein Acetylation by Dot-blot
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3T3-L1 cells treated with 10 μM EX527 (an inhibitor of deacetylase SIRT1) or 1 μM of Trichostatin A (TSA, Wako) and 10 mM of Nicotinamide (NAM, Sigma) were lysed in ETN buffer (1 mM EDTA, 100 mM NaCl, 50 mM Tris-HCl [pH = 8.0]) containing protease inhibitor cocktail. Cell lysates were sonicated and cleared by centrifugation at 12,000 rpm for 20 min at 4°C. Random peptides containing acetyl-lysine or non-acetyl-lysine were dissolved in 2 mM NaOH solution. The dot-blot apparatus (Biorad) was assembled with a 0.2 μm pore size nitrocellulose membrane that was pre-wetted into TBS then adjust the flow valve to open vacuum chamber. Samples were applied onto wells and allow the entire sample to filter through the membrane by gravity flow. The membrane was removed and rinsed with TBS. Immunoassay was conducted following manufacturer’s instruction and using specific antibodies against pan-acetyl-lysine (ICP0380, ImmuneChem), or SICS antisera from #37 to #44, respectively.
9
Telomere, Alu, and TSQ1 Detection
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DNA was purified and eluted using the PCR cleanup kit (Qiagen). DNA was loaded on Hybond N+ membrane using a dot blot apparatus (Bio-Rad), and telomeric sequences were detected using a randomly labeled TeloC probe (Grolimund et al. 2013 (link)). For the detection of Alu sequences, membranes were stripped and reprobed with a 5′ end 32P-labeled oligonucleotide probe specific for Alu DNA (5′-GTGATCCGCCCGCCTCGGCCTCCCAAAGTG-3′).
For the detection of TSQ1 sequences, restriction-digested genomic DNA was pipetted on a Hybond N+ membrane (Amersham), UV cross-linked, and sequentially treated with denaturation and neutralization buffers for 15 min each. Church mix prehybridized membrane was incubated with 5′ end 32P-labeled oligonucleotide TSQ1 probe (5′-CCGCAACCGCAACCGCAA-3′) for the detection of incorporated TSQ1 repeat sequences.
For the detection of TSQ1 sequences, restriction-digested genomic DNA was pipetted on a Hybond N+ membrane (Amersham), UV cross-linked, and sequentially treated with denaturation and neutralization buffers for 15 min each. Church mix prehybridized membrane was incubated with 5′ end 32P-labeled oligonucleotide TSQ1 probe (5′-CCGCAACCGCAACCGCAA-3′) for the detection of incorporated TSQ1 repeat sequences.
10
Quantification of α-Synuclein Fibrils in Cell Culture
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Culture media was conditioned for last 24 h of experiment. Media was removed from cells and centrifuged at 1000 × g for 5 min to remove floating cells. Cells were harvested and lysed in 1% (v/v) TX-100 in PBS and total protein content of well calculated using the BCA assay. Media was applied to PROTRAN nitrocellulose (Perkin Elmer) in triplicate using a dot blot apparatus (Bio-Rad). A PFF standard curve was also applied with serial dilution of PFF in culture media (0–30 ng/ml). Following two PBS washes, the membrane was blocked with BlockACE (BioRad) and incubated overnight at 4°C with anti-α-syn filament antibody (abcam, ab209538). Following incubation with anti-rabbit HRP-conjugated secondary antibody, membranes were incubated with Immobilon Luminata Forte enhanced chemiluminescence (Merck) and images acquired and quantified using Image Lab software (BioRad). Dot density was converted to ng/ml using the PFF standard curve and normalized to protein content of the well. The mean of each triplicate was calculated and expressed as ng fibrils/mg protein.
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