Mini blot module

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Mini Blot Module is a compact and efficient lab equipment designed for protein transfer and western blotting applications. It facilitates the transfer of proteins from a gel to a membrane for further analysis. The module provides a simple and reliable solution for researchers requiring a streamlined blotting process.

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Lab products found in correlation

Mini gel tank, by Thermo Fisher Scientific (6 mentions) Dc protein assay kit, by Bio-Rad (4 mentions) Odyssey clx imager, by LI COR (4 mentions) β actin, by Merck Group (4 mentions) Image studio lite software, by LI COR (4 mentions) Odyssey blocking buffer, by LI COR (3 mentions) Odyssey clx infrared imaging system, by LI COR (3 mentions) Chemidoc imaging system, by Bio-Rad (3 mentions) Nupage bis tris gel, by Thermo Fisher Scientific (3 mentions) Bolt 4 12 bis tris plus gel, by Thermo Fisher Scientific (3 mentions) Bolt transfer buffer, by Thermo Fisher Scientific (3 mentions) Hybond, by Cytiva (2 mentions) Anti myc 9b11, by Cell Signaling Technology (2 mentions) Anti gfp, by Roche (2 mentions) Near infrared fluorescence, by LI COR (2 mentions) Iblot 2 gel transfer device, by Thermo Fisher Scientific (2 mentions) Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions) Fastprep 24 5g instrument, by MP Biomedicals (2 mentions) Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions) Las 4000, by Cytiva (2 mentions)

Spelling variants of this product

Bolt Mini Blot Module (8 citations) XCell II Mini-Cell Blot Module kit (4 citations) Mini-blot (3 citations) Mini Gel Tank and Blot Module Set (2 citations) Bolt™ Mini Blot Module and Mini Gel Tank (2 citations)

32 protocols using mini blot module

Phosphorylation of Aurora Kinase A

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Purified Aurora (50 ng) was run on a 4–20% Novex WedgeWell Tris-Glycine gel (Life Technologies). Protein was transferred to a 0.45 um pore size pure nitrocellulose membrane (VWR International) running at 10 V for 1 hour using a Mini Blot Module (Life Technologies). The membrane was blocked for 1 hour at room temperature in 5% BSA (Fisher Scientific) dissolved in PBST. The membrane was incubated with a 1:10,000 dilution of phospho-AurA (Thr288) Rabbit AB primary antibody (Cell Signaling Technology) for 1 hour at room temperature. The membrane was washed in PBST and then blocked in 5% BSA in PBST for one hour. The membrane was incubated with a 1:10,000 dilution of peroxidase-conjugated AffiniPure Donkey Anti-rabbit IgG (Jackson ImmunoReserach Laboratories Inc) for 45 minutes at room temperature. The membrane was washed with PBST. Supersignal Chemiluminescent Substrate (ThermoScientific) was added to the membrane and the membrane was visualized on a myECL imager (ThermoScientific).

Cyphers S., Ruff E.F., Behr J., Chodera J.D, & Levinson N.M. (2017). A water-mediated allosteric network governs activation of Aurora kinase A. Nature chemical biology, 13(4), 402-408.

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Protein Extraction and Immunoblotting

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Cells were lysed in RIPA buffer (10mM Tris-HCl [pH 8.0], 150mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100) with protease and phosphatase inhibitors (Roche). Protein concentrations were measured using the DC Protein Assay Kit (Bio-Rad). SDS–PAGE and protein transfer were performed using Mini Gel Tank and Mini Blot Module (Life Technologies). Immunoblotting was detected using near-infrared fluorescence and the Odyssey CLx imager (LI-COR). Quantitative analysis of immunoblots was performed using Image Studio Lite software (LI-COR). The following primary antibodies were used from Cell Signaling Technologies: Phospho-PAK1 (Ser144)/PAK2 (Ser141) (Cat. No. 2606S), Phospho-PAK1 (Ser199/204)/PAK2 (Ser192/197) (Cat. No. 2605T), Phospho-PAK1 (Thr423)/PAK2 (Thr402) (Cat. No. 2601T), Phospho-PAK2 (Ser20) (Cat. No. 2607T). Additional antibodies: Pak1 (Santa Cruz, sc-882), p-Erk1/2 (CST, 4370), Erk1/2 (CST, 4695) and β-actin (Sigma, A1978), HA (Thermo, 26183).

Lee S.W, & Commisso C. (2020). Rac1 and EGFR cooperate to activate Pak in response to nutrient stress. Biochemical and biophysical research communications, 533(3), 437-441.

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Immunoblotting Analysis of EGFR Signaling

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Cells were lysed in RIPA buffer (10mM Tris-HCl [pH 8.0], 150mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100) with protease and phosphatase inhibitors (Roche). Protein concentrations were measured using the DC Protein Assay Kit (Bio-Rad). SDS–PAGE and protein transfer were performed using Mini Gel Tank and Mini Blot Module (Life Technologies). Immunoblotting was detected using near-infrared fluorescence and the Odyssey CLx imager (LI-COR). Quantitative analysis of immunoblots was performed using Image Studio Lite software (LI-COR). The following primary antibodies were used: p-EGFR (Y1068) (CST, 3777), pEGFR (T669) (Abcam, ab227017), EGFR (CST, 4267), p-Erk1/2 (T202/Y204) (CST, 4370), Erk1/2 (CST, 4695), p-Akt (S473) (CST, 4060), pan-Akt (CST, 4691), K-Ras (sc-30) and β-actin (Sigma, A1978). At least three independent experiments were performed in all cases.

Lee S.W, & Commisso C. (2020). Metabolic regulation of EGFR effector and feedback signaling in pancreatic cancer cells requires K-Ras. Biochemical and biophysical research communications, 533(3), 424-428.

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Aβ Oligomerization Dynamics Elucidated

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Aβ_1-42 (100 μM) alone and in combination with different molar ratios
of Aβ_8-20 (1:1; 1:5; and 1:10) was incubated
at 4 °C for 48 h to form Aβ oligomers. After incubation,
the amount and size of Aβ aggregates were determined by Western
blot analysis. A volume of 25 μL of each unheated sample was
loaded onto a precast Bis-Tris gel (Bolt 4–12%, Life Technologies)
with 2-morpholin-4-yl ethanesulfonic acid. Samples were transferred
onto a nitrocellulose membrane (0.2 mm, Hybond ECL, Amersham Italia)
by using a wet transfer unit Mini Blot Module (Life Technologies).
Membranes were blocked in Odyssey blocking buffer (Li-COR Biosciences)
and incubated at 4 °C overnight with anti-Aβ N-terminal
1–16 mouse monoclonal antibody 6E10 (1:500) (BioLegend). A
secondary goat anti-mouse antibody labeled with infrared dye (1:20.000)
was used at room temperature for 45 min. Hybridization signals were
detected with the Odyssey CLx infrared imaging system (LI-COR Biosciences,
Lincoln, NE).

Zimbone S., Giuffrida M.L., Sabatino G., Di Natale G., Tosto R., Consoli G.M., Milardi D., Pappalardo G, & Sciacca M.F. (2023). Aβ8-20 Fragment as an Anti-Fibrillogenic and Neuroprotective Agent: Advancing toward Efficient Alzheimer’s Disease Treatment. ACS Chemical Neuroscience, 14(6), 1126-1136.

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Caffeine, KCl, and H2O2 Stress Assay

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Cells were treated with 14mM caffeine, 1M KCl, 1mM H2O2 for 16 hours, with fresh H2O2 added every 2 hours to compensate for its decomposition. Protein samples were prepared essentially as previously detailed³. Briefly, a 10 ml culture of log phase cells was harvested, resuspended in 1 ml of cold water and transferred to an eppendorf tube. Cells were pelleted and resuspended in 775μl water. 150μl 1.85M NaOH and β-mercaptoethanol 7.5% were added and the samples incubated on ice for 15 minutes. 150μl 55% TCA was added and incubated for a further 10 minutes on ice. Samples were centrifuged at 16,800xg for 10 minutes and pellets were resuspended in HU-buffer (8M urea, 5% SDS, 200mM Tris pH 6·8, 1mM EDTA, with bromo-phenol blue, 1·5 DTT).
Samples were run on a 4-12% NuPAGE Bis-Tris gel (ThermoFisher) and blotted using the MiniBlot Module (Life Technologies) 20V for 1 hour. Western blotting detection was performed using anti-myc 9B11 (Cell Signalling) 1:1000, anti GFP (Roche) 1:1000 and anti-Mouse IgG (whole molecule)–Peroxidase antibody (Sigma) 1:10000. Gels were visualised using the ChemiDoc imaging system (BioRad) and analysed with ImageJ. Where quantified, levels of full-length (FL) Epe1 and truncated tEpe1 were normalised to no treatment and adjusted relative to the α-tubulin loading control. Resulting numbers are an average of 3 biological replicates.

Yaseen I., White S.A., Torres-Garcia S., Spanos C., Lafos M., Gaberdiel E., Yeboah R., Karoui M.E., Rappsilber J., Pidoux A.L, & Allshire R.C. (2022). Proteasome-dependent truncation of the negative heterochromatin regulator Epe1 mediates antifungal resistance. Nature structural & molecular biology, 29(8), 745-758.

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Quantifying Amyloid-β Aggregation by Western Blot

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After incubation, the amount
and size of Aβ aggregates were determined by western blot analysis.
Unheated samples (25 μL) were loaded onto precast Bis–Tris
gel (Bolt 4–12%, Life Technologies) with 2-morpholin-4-ylethanesulfonic
acid (MES). Samples were transferred onto a nitrocellulose membrane
(0.2 mm, Hybond ECL, Amersham Italia) by a wet transfer unit (Mini
Blot Module, Life Technologies). Membranes were blocked in the Odyssey
blocking buffer (Li-COR Biosciences) and incubated at 4 °C overnight
with the mouse monoclonal anti-amyloid-β antibody against N-terminal
1–16 peptide (1:1000) (6E10, Covance). Goat anti-mouse secondary
antibodies labeled with IR dye 800 (1:25,000) were used at RT for
45 min. Hybridization signals were detected with the Odyssey CLx Infrared
Imaging System (LI-COR Biosciences Lincoln, Nebraska, USA), and densitometric
analysis was performed by the use of the Image Studio software.

Sciacca M.F., Naletova I., Giuffrida M.L, & Attanasio F. (2022). Semax, a Synthetic Regulatory Peptide, Affects Copper-Induced Abeta Aggregation and Amyloid Formation in Artificial Membrane Models. ACS Chemical Neuroscience, 13(4), 486-496.

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Aβ Oligomers Formation and Analysis

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A stock aliquot of 5 mM Aβ
previously dissolved in DMSO was diluted in DMEM F-12 without Phenol
Red, and two different samples at 20 μM and 50 μM concentrations
were prepared. From each solution, two sets of samples were obtained
by combining Aβ₄₂ with KLVFF peptide, GPGKLVFF, or
compound 5 with a molar ratio Aβ/peptide of 1:5.
All the samples, including Aβ alone at the two concentrations
used, were incubated at 4 °C for 48 h to form Aβ oligomers.
After incubation, the amount and size of Aβ aggregates were
determined by Western blot analysis. A volume of 25 μL of each
unheated sample was loaded onto a precast Bis-Tris gel (Bolt 4–12%,
Life Technologies) with 2-morpholin-4-yl ethanesulfonic acid (MES).
Samples were transferred onto a nitrocellulose membrane (0.2 mm, Hybond
ECL, Amersham Italia) by using a wet transfer unit Mini Blot Module
(Life Technologies). Membranes were blocked in Odyssey blocking buffer
(Li-COR Biosciences) and incubated at 4 °C overnight with mouse
monoclonal anti-amyloid-β antibody against N-terminal 1–16
peptide (1:1000) (Covance). A secondary goat anti-mouse antibody labeled
with IR dye 800 (1:25 000) was used at rt for 45 min. Hybridization
signals were detected with the Odyssey CLx infrared imaging system
(LI-COR Biosciences, Lincoln, NE).

Consoli G.M., Tosto R., Baglieri A., Petralia S., Campagna T., Di Natale G., Zimbone S., Giuffrida M.L, & Pappalardo G. (2021). Novel Peptide-Calix[4]arene Conjugate Inhibits Aβ Aggregation and Rescues Neurons from Aβ’s Oligomers Cytotoxicity In Vitro. ACS Chemical Neuroscience, 12(8), 1449-1462.

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GR Protein Expression Quantification

Cited 2 times

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Immunoblotting was carried out following established protocols [2 (link),84 (link)]. Antibody concentrations and loaded total proteins amounts (15 μg) of male and female PNL and nuc fractions were optimized to fit the linear range of signal detection. Protein expression was evaluated in GD14, GD16 and GD18, male and female, PNL and nuc fractions (n ≥ 5). Transfer was done onto Immobilon-FL Transfer Membrane (cat: IPFL07810, Millipore) using a Mini Blot Module (cat: B1000, Life Technologies). Blots were incubated in in LI-COR Blocking Buffer (cat: 927-40000, LI-COR) overnight at +4° C. Membranes were then incubated with anti-GR antibody (cat: sc-1004, Santa Cruz Biotechnology) diluted 1:1,000 in PBS-T (10x PBS (cat: 70011-044, Life Technologies) diluted in deionized water, 1% v/v Tween-20) for 3 hours at room temperature. An immunofluorescent secondary antibody was applied (IRDye 680 Donkey anti-Rabbit IgG (cat: 926-68073) diluted 1:10,000 in PBS-T) for 45 minutes at room temperature without light exposure. Imaging of the immunoblots was conducted using a two-channel infrared detection Odyssey Infrared Imaging System (cat: LIC-8201-00, LI-COR) and quantified using Image Studio version 5.0 (LI-COR Biosciences). Protein detected by immunofluorescence was corrected to the total protein as quantified by Coomassie staining [65 (link)].

Caldwell K.K., Hafez A., Solomon E., Cunningham M, & Allan A.M. (2017). Arsenic exposure during embryonic development alters the expression of the Long noncoding RNA Growth arrest specific-5 (Gas5) in a sex-dependent manner. Neurotoxicology and teratology, 66, 102-112.

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Fibroblast Protein Extraction and Analysis

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Primary fibroblasts were lysed with protein lysis buffer containing 10 mM Tris–HCl, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10% glycerol, 10% proteinase inhibitors cocktail (Roche, 11 836 153 001) and 1 mM DTT. Total cell protein was obtained after 30 min of centrifugation (16,000 rpm). Protein concentration was determined with PierceTM BCA protein assay kit (Thermo Scientific, 23227) according to manufacturer’s instructions by reading absorbance at 562 nm with Epoch microplate spectrophotometer (BioTek). Protein volumes were adjusted to bring equal amounts on gel, mixed with BoltTM LDS sample buffer (Life Technologies, B0007) and BoltTM sample reducing agent (Life Technologies, B0009) according to manufacturer’s instructions. Afterwards, the samples were denaturated at 70 °C for 10 min and transferred to Bolt 4–12% Bis–Tris Plus Gel (Life Technologies, NW04120BOX). Samples were run at 165 V for 45 min in BoltTM MOPS SDS running buffer (Life Technologies, B0001). Proteins were transferred to the nitrocellulose membrane (Life Technologies, LC2000) at 10 V for 1 h and 15 min using Mini Blot Module (Life Technologies, B1000) and Bolt transfer buffer (Life technologies, BT0006) according to manufacturer’s instructions.

Lessel I., Chen M.J., Lüttgen S., Arndt F., Fuchs S., Meien S., Thiele H., Jones J.R., Shaw B.R., Crossman D.K., Nürnberg P., Korf B.R., Kubisch C, & Lessel D. (2020). Two novel cases further expand the phenotype of TOR1AIP1-associated nuclear envelopathies. Human Genetics, 139(4), 483-498.

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Protein Extraction and Western Blot Analysis

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Cells were lysed in RIPA buffer (10mM Tris-HCl [pH 8.0], 150mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100) with protease and phosphatase inhibitors (Roche). Protein concentrations were measured using the DC Protein Assay Kit (Bio-Rad). SDS–PAGE and protein transfer were performed using Mini Gel Tank and Mini Blot Module (Life Technologies). Immunoblotting was detected using near-infrared fluorescence (LI-COR) and the Odyssey CLx imager (LI-COR). Quantitative analysis of immunoblots was performed using Image Studio Lite software (LI-COR). The following primary antibodies were used: EREG (1:1000) [Cell Signaling Technologies (CST), 12048], HB-EGF (1:10000) (Abcam, ab185555), p-EGFR (Y1068) (1:1000) (CST, 3777), EGFR (1:1000) (CST, 4267), p-PAK1 (S199/204)/p-PAK2 (S192/197) (1:500) (CST, 2605), PAK1 (1:300) (Santa Cruz, sc-882), p-Erk1/2 (T202/Y204) (1:2000) (CST, 4370), Erk1/2 (1:1000) (CST, 4695), p-Akt (S473) (1:1500) (CST, 4060), pan-Akt (1:1000) (CST, 4691), α-tubulin (1:10000) (Sigma, T6074), and β-actin (1:20000) (Sigma, A1978). To analyze phosphorylation of proteins, membranes were probed with phospho-specific antibodies first, stripped with NewBlot IR Stripping Buffer (LI-COR) and then reprobed with pan antibodies.

Lee S.W., Zhang Y., Jung M., Cruz N., Alas B, & Commisso C. (2019). EGFR/Pak Signaling Selectively Regulates Glutamine Deprivation-Induced Macropinocytosis. Developmental cell, 50(3), 381-392.e5.

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