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Carl Roth GmbH sds gel loading buffer
Sds Gel Loading Buffer, supplied by Carl Roth GmbH, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/sds+loading+buffer/sds+gel+loading+buffer/pmc03365809-201-19-24
Average 90 stars, based on 1 article reviews
sds gel loading buffer - by Bioz Stars, 2026-09
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Article Title: Assessing the Molecular Targets and Mode of Action of Furanone C-30 on Pseudomonas aeruginosa Quorum Sensing
Article Snippet: Sample (loading) buffer for SDS–PAGE was purchased from Carl Roth (Karlsruhe, Germany).

Article Title: REVERBA couples the circadian clock to Leydig cell steroidogenesis.
Article Snippet: Funding information The Provincial Secretariat for Higher Education and Scientific Research, Grant/Award Number: APV2708; Ministry of Science, Technological Development and Innovation of the Republic of Serbia, Grant/Award Numbers: 451-03-47/2023-01/200125, CeRES grant Abstract The involvement of the molecular clock in regulating cell physiological processes on a specific time scale is a recognized concept, yet its specific impact on optimizing androgen production in Leydig cells has been unclear.. This study aimed to confirm the role of the REVERBA (NR1D1) gene in controlling the transcription of key genes related to Leydig cell steroid production.. We investigated daily variations by collecting Leydig cells from rats at various times within a 24-h period.

Article Title: PLOD2 Is a Prognostic Marker in Glioblastoma That Modulates the Immune Microenvironment and Tumor Progression
Article Snippet: Cell debris was removed by centrifugation and the lysates were incubated with an SDS-Loading buffer containing 4% glycerin, 0.8% SDS, 1.6% beta-mercaptoethanol and 0.04% bromophenol blue (all from Carl Roth).

Article Title: Assessing the Molecular Targets and Mode of Action of Furanone C-30 on Pseudomonas aeruginosa Quorum Sensing
Article Snippet: Sample (loading) buffer for SDS–PAGE was purchased from Carl Roth (Karlsruhe, Germany).


SDS Page:

Article Title: Development of novel genetically-encoded fluorescent probes to track ceramides during phagocytosis
Article Snippet: Protein concentration was quantitated using RotiQuant kit (Carl Roth). .. Lysates were diluted in the SDS Loading Buffer (Roti-Load, Carl Roth) and heated at 95°C for 5 min. 15-30 μg protein were loaded and separated by SDS-PAGE in 4–12% SurePAGE 12-well pre-cast gels (Genscript). .. Proteins were transferred onto PVDF membranes using iBlot or iBlot2 system (Thermo).

Article Title: Effects of a Multimerized Recombinant Autoantibody Against Amyloid-β.
Article Snippet: Alzheimer’s disease (AD) is the most common neurodegenerative disease; thus, the search for a cure or causal therapy has become necessary.. Despite intense research on this topic in recent decades, there is no curative therapy up today, and also no disease-modifying treatment has been approved.. As promising approach passive immunization strategies have thereby come forth.

MTT Assay:

Article Title: Iron(III)-salophene catalyzes redox cycles that induce phospholipid peroxidation and deplete cancer cells of ferroptosis-protecting cofactors.
Article Snippet: Ethanol (Fisher Scientific, #10048291; 16.7 %) and staurosporine (Sigma Aldrich, #S4400; 1 μM) were used as controls. .. To each well, 20 μL MTT (5 mg/mL in phosphate-buffered saline (PBS) pH 7.4, sterile filtered) was added and the incubation continued for 1.5 h. Cells were lysed and MTT was solubilized by adding 100 μL SDS buffer (Carl Roth, #2326.2; 10 % SDS in 20 mM HCl, pH 4.5) with shaking in the dark for 20 h. Absorbance was measured at 570 nm using a SpectraMAX iD3 spectrometer (Molecular Devices, San José, CA) and normalized to vehicle (100 % cell viability) and ethanol control (0 % cell viability). .. MCF-12A cells (1 × 104) and MDA-MB-231 cells (1 × 104) were seeded in 6-well plates (Fisher Scientific, #10380291) in 2 mL of culture medium for 24 h, then the medium was replaced every 2 days with fresh culture medium containing vehicle (0.1 % DMSO) or test compounds.

Saline:

Article Title: Iron(III)-salophene catalyzes redox cycles that induce phospholipid peroxidation and deplete cancer cells of ferroptosis-protecting cofactors.
Article Snippet: Ethanol (Fisher Scientific, #10048291; 16.7 %) and staurosporine (Sigma Aldrich, #S4400; 1 μM) were used as controls. .. To each well, 20 μL MTT (5 mg/mL in phosphate-buffered saline (PBS) pH 7.4, sterile filtered) was added and the incubation continued for 1.5 h. Cells were lysed and MTT was solubilized by adding 100 μL SDS buffer (Carl Roth, #2326.2; 10 % SDS in 20 mM HCl, pH 4.5) with shaking in the dark for 20 h. Absorbance was measured at 570 nm using a SpectraMAX iD3 spectrometer (Molecular Devices, San José, CA) and normalized to vehicle (100 % cell viability) and ethanol control (0 % cell viability). .. MCF-12A cells (1 × 104) and MDA-MB-231 cells (1 × 104) were seeded in 6-well plates (Fisher Scientific, #10380291) in 2 mL of culture medium for 24 h, then the medium was replaced every 2 days with fresh culture medium containing vehicle (0.1 % DMSO) or test compounds.

Sterility:

Article Title: Iron(III)-salophene catalyzes redox cycles that induce phospholipid peroxidation and deplete cancer cells of ferroptosis-protecting cofactors.
Article Snippet: Ethanol (Fisher Scientific, #10048291; 16.7 %) and staurosporine (Sigma Aldrich, #S4400; 1 μM) were used as controls. .. To each well, 20 μL MTT (5 mg/mL in phosphate-buffered saline (PBS) pH 7.4, sterile filtered) was added and the incubation continued for 1.5 h. Cells were lysed and MTT was solubilized by adding 100 μL SDS buffer (Carl Roth, #2326.2; 10 % SDS in 20 mM HCl, pH 4.5) with shaking in the dark for 20 h. Absorbance was measured at 570 nm using a SpectraMAX iD3 spectrometer (Molecular Devices, San José, CA) and normalized to vehicle (100 % cell viability) and ethanol control (0 % cell viability). .. MCF-12A cells (1 × 104) and MDA-MB-231 cells (1 × 104) were seeded in 6-well plates (Fisher Scientific, #10380291) in 2 mL of culture medium for 24 h, then the medium was replaced every 2 days with fresh culture medium containing vehicle (0.1 % DMSO) or test compounds.

Incubation:

Article Title: Iron(III)-salophene catalyzes redox cycles that induce phospholipid peroxidation and deplete cancer cells of ferroptosis-protecting cofactors.
Article Snippet: Ethanol (Fisher Scientific, #10048291; 16.7 %) and staurosporine (Sigma Aldrich, #S4400; 1 μM) were used as controls. .. To each well, 20 μL MTT (5 mg/mL in phosphate-buffered saline (PBS) pH 7.4, sterile filtered) was added and the incubation continued for 1.5 h. Cells were lysed and MTT was solubilized by adding 100 μL SDS buffer (Carl Roth, #2326.2; 10 % SDS in 20 mM HCl, pH 4.5) with shaking in the dark for 20 h. Absorbance was measured at 570 nm using a SpectraMAX iD3 spectrometer (Molecular Devices, San José, CA) and normalized to vehicle (100 % cell viability) and ethanol control (0 % cell viability). .. MCF-12A cells (1 × 104) and MDA-MB-231 cells (1 × 104) were seeded in 6-well plates (Fisher Scientific, #10380291) in 2 mL of culture medium for 24 h, then the medium was replaced every 2 days with fresh culture medium containing vehicle (0.1 % DMSO) or test compounds.

Control:

Article Title: Iron(III)-salophene catalyzes redox cycles that induce phospholipid peroxidation and deplete cancer cells of ferroptosis-protecting cofactors.
Article Snippet: Ethanol (Fisher Scientific, #10048291; 16.7 %) and staurosporine (Sigma Aldrich, #S4400; 1 μM) were used as controls. .. To each well, 20 μL MTT (5 mg/mL in phosphate-buffered saline (PBS) pH 7.4, sterile filtered) was added and the incubation continued for 1.5 h. Cells were lysed and MTT was solubilized by adding 100 μL SDS buffer (Carl Roth, #2326.2; 10 % SDS in 20 mM HCl, pH 4.5) with shaking in the dark for 20 h. Absorbance was measured at 570 nm using a SpectraMAX iD3 spectrometer (Molecular Devices, San José, CA) and normalized to vehicle (100 % cell viability) and ethanol control (0 % cell viability). .. MCF-12A cells (1 × 104) and MDA-MB-231 cells (1 × 104) were seeded in 6-well plates (Fisher Scientific, #10380291) in 2 mL of culture medium for 24 h, then the medium was replaced every 2 days with fresh culture medium containing vehicle (0.1 % DMSO) or test compounds.

Glycoproteomics:

Article Title: Effects of a Multimerized Recombinant Autoantibody Against Amyloid-β.
Article Snippet: Alzheimer’s disease (AD) is the most common neurodegenerative disease; thus, the search for a cure or causal therapy has become necessary.. Despite intense research on this topic in recent decades, there is no curative therapy up today, and also no disease-modifying treatment has been approved.. As promising approach passive immunization strategies have thereby come forth.

Extraction:

Article Title: Effects of a Multimerized Recombinant Autoantibody Against Amyloid-β.
Article Snippet: Alzheimer’s disease (AD) is the most common neurodegenerative disease; thus, the search for a cure or causal therapy has become necessary.. Despite intense research on this topic in recent decades, there is no curative therapy up today, and also no disease-modifying treatment has been approved.. As promising approach passive immunization strategies have thereby come forth.

Recombinant:

Article Title: Effects of a Multimerized Recombinant Autoantibody Against Amyloid-β.
Article Snippet: Alzheimer’s disease (AD) is the most common neurodegenerative disease; thus, the search for a cure or causal therapy has become necessary.. Despite intense research on this topic in recent decades, there is no curative therapy up today, and also no disease-modifying treatment has been approved.. As promising approach passive immunization strategies have thereby come forth.



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a , Domain architecture of RAP80 and ARISC constructs. FL, full-length; SIM, small ubiquitin-like modifier (SUMO)-interacting motif; UIM, ubiquitin-interacting motif; AIR, Abraxas1-interacting region; ZnF, zinc finger; MPN, Mpr1, Pad1 N-terminal; CC, coiled coil; UEV, ubiquitin E2 variant; vWFA, von Willebrand factor type A ( left ). Schematics of indicated complexes ( right ). b <t>,</t> <t>SDS-PAGE</t> analysis of ARISC, ARISC–RAP80, and ARISC–RAP80 AIR. c , K63-linked ubiquitin chains (1 µM) were incubated with ARISC or ARISC–RAP80 (5 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and silver staining. Data are representative of two independent experiments. d , K63-Ub2, -Ub4, and - Ub7 chains (1 µM) were incubated with ARISC, ARISC–RAP80, or ARISC–RAP80 AIR (5 nM) for the indicated time points. Cleavage activity was analysed as in c . Data are representative of three independent experiments. e , Schematics ( left ) and SDS-PAGE analysis ( right ) of indicated complexes. dStrepII, double StrepII tag. * indicates Abraxas1 degradation product. f , Alexa-Fluor 488 (AF488) labelled distally (AF488- Cys Ub4 K63R ) blocked K63-Ub4 chains (1.5 µM) were incubated with ARISC–RAP80, ARISC–RAP80 ΔUIMs, or ARISC–RAP80 ΔZnF (10 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and fluorescence scanning ( left ; see Methods ). The disappearance of the K63-Ub4 parent band was quantified using densitometry, and plotted as fraction of substrate consumed (%). Data points are mean ± SEM of two independent experiments ( right ). g , Cyclical and linear K63-Ub5 chains (2 µM) were incubated with ARISC or ARISC–RAP80 (10 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and Oriole staining. Data are representative of two independent experiments. Ub, ubiquitin; DUB, deubiquitylating enzyme. * indicates lower molecular weight ubiquitin species.
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a , Domain architecture of RAP80 and ARISC constructs. FL, full-length; SIM, small ubiquitin-like modifier (SUMO)-interacting motif; UIM, ubiquitin-interacting motif; AIR, Abraxas1-interacting region; ZnF, zinc finger; MPN, Mpr1, Pad1 N-terminal; CC, coiled coil; UEV, ubiquitin E2 variant; vWFA, von Willebrand factor type A ( left ). Schematics of indicated complexes ( right ). b <t>,</t> <t>SDS-PAGE</t> analysis of ARISC, ARISC–RAP80, and ARISC–RAP80 AIR. c , K63-linked ubiquitin chains (1 µM) were incubated with ARISC or ARISC–RAP80 (5 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and silver staining. Data are representative of two independent experiments. d , K63-Ub2, -Ub4, and - Ub7 chains (1 µM) were incubated with ARISC, ARISC–RAP80, or ARISC–RAP80 AIR (5 nM) for the indicated time points. Cleavage activity was analysed as in c . Data are representative of three independent experiments. e , Schematics ( left ) and SDS-PAGE analysis ( right ) of indicated complexes. dStrepII, double StrepII tag. * indicates Abraxas1 degradation product. f , Alexa-Fluor 488 (AF488) labelled distally (AF488- Cys Ub4 K63R ) blocked K63-Ub4 chains (1.5 µM) were incubated with ARISC–RAP80, ARISC–RAP80 ΔUIMs, or ARISC–RAP80 ΔZnF (10 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and fluorescence scanning ( left ; see Methods ). The disappearance of the K63-Ub4 parent band was quantified using densitometry, and plotted as fraction of substrate consumed (%). Data points are mean ± SEM of two independent experiments ( right ). g , Cyclical and linear K63-Ub5 chains (2 µM) were incubated with ARISC or ARISC–RAP80 (10 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and Oriole staining. Data are representative of two independent experiments. Ub, ubiquitin; DUB, deubiquitylating enzyme. * indicates lower molecular weight ubiquitin species.
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a , Domain architecture of RAP80 and ARISC constructs. FL, full-length; SIM, small ubiquitin-like modifier (SUMO)-interacting motif; UIM, ubiquitin-interacting motif; AIR, Abraxas1-interacting region; ZnF, zinc finger; MPN, Mpr1, Pad1 N-terminal; CC, coiled coil; UEV, ubiquitin E2 variant; vWFA, von Willebrand factor type A ( left ). Schematics of indicated complexes ( right ). b <t>,</t> <t>SDS-PAGE</t> analysis of ARISC, ARISC–RAP80, and ARISC–RAP80 AIR. c , K63-linked ubiquitin chains (1 µM) were incubated with ARISC or ARISC–RAP80 (5 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and silver staining. Data are representative of two independent experiments. d , K63-Ub2, -Ub4, and - Ub7 chains (1 µM) were incubated with ARISC, ARISC–RAP80, or ARISC–RAP80 AIR (5 nM) for the indicated time points. Cleavage activity was analysed as in c . Data are representative of three independent experiments. e , Schematics ( left ) and SDS-PAGE analysis ( right ) of indicated complexes. dStrepII, double StrepII tag. * indicates Abraxas1 degradation product. f , Alexa-Fluor 488 (AF488) labelled distally (AF488- Cys Ub4 K63R ) blocked K63-Ub4 chains (1.5 µM) were incubated with ARISC–RAP80, ARISC–RAP80 ΔUIMs, or ARISC–RAP80 ΔZnF (10 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and fluorescence scanning ( left ; see Methods ). The disappearance of the K63-Ub4 parent band was quantified using densitometry, and plotted as fraction of substrate consumed (%). Data points are mean ± SEM of two independent experiments ( right ). g , Cyclical and linear K63-Ub5 chains (2 µM) were incubated with ARISC or ARISC–RAP80 (10 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and Oriole staining. Data are representative of two independent experiments. Ub, ubiquitin; DUB, deubiquitylating enzyme. * indicates lower molecular weight ubiquitin species.
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a , Domain architecture of RAP80 and ARISC constructs. FL, full-length; SIM, small ubiquitin-like modifier (SUMO)-interacting motif; UIM, ubiquitin-interacting motif; AIR, Abraxas1-interacting region; ZnF, zinc finger; MPN, Mpr1, Pad1 N-terminal; CC, coiled coil; UEV, ubiquitin E2 variant; vWFA, von Willebrand factor type A ( left ). Schematics of indicated complexes ( right ). b <t>,</t> <t>SDS-PAGE</t> analysis of ARISC, ARISC–RAP80, and ARISC–RAP80 AIR. c , K63-linked ubiquitin chains (1 µM) were incubated with ARISC or ARISC–RAP80 (5 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and silver staining. Data are representative of two independent experiments. d , K63-Ub2, -Ub4, and - Ub7 chains (1 µM) were incubated with ARISC, ARISC–RAP80, or ARISC–RAP80 AIR (5 nM) for the indicated time points. Cleavage activity was analysed as in c . Data are representative of three independent experiments. e , Schematics ( left ) and SDS-PAGE analysis ( right ) of indicated complexes. dStrepII, double StrepII tag. * indicates Abraxas1 degradation product. f , Alexa-Fluor 488 (AF488) labelled distally (AF488- Cys Ub4 K63R ) blocked K63-Ub4 chains (1.5 µM) were incubated with ARISC–RAP80, ARISC–RAP80 ΔUIMs, or ARISC–RAP80 ΔZnF (10 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and fluorescence scanning ( left ; see Methods ). The disappearance of the K63-Ub4 parent band was quantified using densitometry, and plotted as fraction of substrate consumed (%). Data points are mean ± SEM of two independent experiments ( right ). g , Cyclical and linear K63-Ub5 chains (2 µM) were incubated with ARISC or ARISC–RAP80 (10 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and Oriole staining. Data are representative of two independent experiments. Ub, ubiquitin; DUB, deubiquitylating enzyme. * indicates lower molecular weight ubiquitin species.
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Image Search Results


a , Domain architecture of RAP80 and ARISC constructs. FL, full-length; SIM, small ubiquitin-like modifier (SUMO)-interacting motif; UIM, ubiquitin-interacting motif; AIR, Abraxas1-interacting region; ZnF, zinc finger; MPN, Mpr1, Pad1 N-terminal; CC, coiled coil; UEV, ubiquitin E2 variant; vWFA, von Willebrand factor type A ( left ). Schematics of indicated complexes ( right ). b , SDS-PAGE analysis of ARISC, ARISC–RAP80, and ARISC–RAP80 AIR. c , K63-linked ubiquitin chains (1 µM) were incubated with ARISC or ARISC–RAP80 (5 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and silver staining. Data are representative of two independent experiments. d , K63-Ub2, -Ub4, and - Ub7 chains (1 µM) were incubated with ARISC, ARISC–RAP80, or ARISC–RAP80 AIR (5 nM) for the indicated time points. Cleavage activity was analysed as in c . Data are representative of three independent experiments. e , Schematics ( left ) and SDS-PAGE analysis ( right ) of indicated complexes. dStrepII, double StrepII tag. * indicates Abraxas1 degradation product. f , Alexa-Fluor 488 (AF488) labelled distally (AF488- Cys Ub4 K63R ) blocked K63-Ub4 chains (1.5 µM) were incubated with ARISC–RAP80, ARISC–RAP80 ΔUIMs, or ARISC–RAP80 ΔZnF (10 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and fluorescence scanning ( left ; see Methods ). The disappearance of the K63-Ub4 parent band was quantified using densitometry, and plotted as fraction of substrate consumed (%). Data points are mean ± SEM of two independent experiments ( right ). g , Cyclical and linear K63-Ub5 chains (2 µM) were incubated with ARISC or ARISC–RAP80 (10 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and Oriole staining. Data are representative of two independent experiments. Ub, ubiquitin; DUB, deubiquitylating enzyme. * indicates lower molecular weight ubiquitin species.

Journal: bioRxiv

Article Title: Mechanism of K63-linked polyubiquitin recognition and cleavage by the BRCA1-A complex

doi: 10.64898/2026.06.05.730395

Figure Lengend Snippet: a , Domain architecture of RAP80 and ARISC constructs. FL, full-length; SIM, small ubiquitin-like modifier (SUMO)-interacting motif; UIM, ubiquitin-interacting motif; AIR, Abraxas1-interacting region; ZnF, zinc finger; MPN, Mpr1, Pad1 N-terminal; CC, coiled coil; UEV, ubiquitin E2 variant; vWFA, von Willebrand factor type A ( left ). Schematics of indicated complexes ( right ). b , SDS-PAGE analysis of ARISC, ARISC–RAP80, and ARISC–RAP80 AIR. c , K63-linked ubiquitin chains (1 µM) were incubated with ARISC or ARISC–RAP80 (5 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and silver staining. Data are representative of two independent experiments. d , K63-Ub2, -Ub4, and - Ub7 chains (1 µM) were incubated with ARISC, ARISC–RAP80, or ARISC–RAP80 AIR (5 nM) for the indicated time points. Cleavage activity was analysed as in c . Data are representative of three independent experiments. e , Schematics ( left ) and SDS-PAGE analysis ( right ) of indicated complexes. dStrepII, double StrepII tag. * indicates Abraxas1 degradation product. f , Alexa-Fluor 488 (AF488) labelled distally (AF488- Cys Ub4 K63R ) blocked K63-Ub4 chains (1.5 µM) were incubated with ARISC–RAP80, ARISC–RAP80 ΔUIMs, or ARISC–RAP80 ΔZnF (10 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and fluorescence scanning ( left ; see Methods ). The disappearance of the K63-Ub4 parent band was quantified using densitometry, and plotted as fraction of substrate consumed (%). Data points are mean ± SEM of two independent experiments ( right ). g , Cyclical and linear K63-Ub5 chains (2 µM) were incubated with ARISC or ARISC–RAP80 (10 nM) for the indicated time points. Cleavage activity was analysed by SDS-PAGE and Oriole staining. Data are representative of two independent experiments. Ub, ubiquitin; DUB, deubiquitylating enzyme. * indicates lower molecular weight ubiquitin species.

Article Snippet: Reactions were stopped with the addition of 3 μL 4x SDS-PAGE loading dye [240 mM Tris-HCl pH 6.8, 40% (v/v) glycerol, 8% (w/v) SDS, 0.04% (w/v) bromophenol blue, and 5% (v/v) β-Mercaptoethanol], and products were separated on 4-12% or 12% Nu-PAGE Bis-Tris gels (Invitrogen).

Techniques: Construct, Ubiquitin Proteomics, Variant Assay, SDS Page, Incubation, Activity Assay, Silver Staining, Fluorescence, Staining, Molecular Weight

a , K63-Ub2, -Ub4, and -Ub7 chains (1 µM) were incubated with ARISC WT or the indicated ARISC variants (5 nM) for 60 minutes. Cleavage activity was analysed by SDS-PAGE and silver staining. Data are representative of two independent experiments. b, SDS-PAGE analysis of ARISC(E33A)–RAP80, ARISC(E33A) BRCC36(S98K) –RAP80, ARISC(E33A) Abraxas1(Δ42-55) –RAP80, and ARISC(E33A) BRCC45(ΔLoop) –RAP80. dStrepII, double StrepII tag. * indicates Abraxas1 degradation product. c, Spectral shift assays measuring binding of labelled ARISC(E33A)–RAP80 or the indicated mutant complexes (40 nM) to cyclical K63-Ub6 chains (20 µM-0 µM). Data points are mean ± SEM of two independent experiments carried out in technical duplicates. Dissociation constants (K d ) are indicated; CI, confidence interval. d, Representative images of WT or mutants BRCC36 IRIF in HT-29 cells 4 h post irradiation (10 Gy). Scale bar is 10 µm. e, Western blots showing BRCC36 protein levels in HT-29 cells reconstituted with WT or mutants BRCC36 as indicated (l eft ). Scatter plot showing quantification of the BRCC36 IRIF described in d . Data represent mean ± SEM derived from n ≥ 300 nuclei examined over two independent experiments; p values are indicated, unpaired two-tailed t test ( right ). f, K63-Ub2, -Ub4, and -Ub7 chains (1 µM) were incubated with ARISC WT or ARISC Δ42-55 (Abraxas1 Δ42-55) (5 nM) for up to 60 minutes. Cleavage activity was analysed as in a . Data are representative of two independent experiments. DUB, deubiquitylating enzyme; WT, wild type; Ub, ubiquitin.

Journal: bioRxiv

Article Title: Mechanism of K63-linked polyubiquitin recognition and cleavage by the BRCA1-A complex

doi: 10.64898/2026.06.05.730395

Figure Lengend Snippet: a , K63-Ub2, -Ub4, and -Ub7 chains (1 µM) were incubated with ARISC WT or the indicated ARISC variants (5 nM) for 60 minutes. Cleavage activity was analysed by SDS-PAGE and silver staining. Data are representative of two independent experiments. b, SDS-PAGE analysis of ARISC(E33A)–RAP80, ARISC(E33A) BRCC36(S98K) –RAP80, ARISC(E33A) Abraxas1(Δ42-55) –RAP80, and ARISC(E33A) BRCC45(ΔLoop) –RAP80. dStrepII, double StrepII tag. * indicates Abraxas1 degradation product. c, Spectral shift assays measuring binding of labelled ARISC(E33A)–RAP80 or the indicated mutant complexes (40 nM) to cyclical K63-Ub6 chains (20 µM-0 µM). Data points are mean ± SEM of two independent experiments carried out in technical duplicates. Dissociation constants (K d ) are indicated; CI, confidence interval. d, Representative images of WT or mutants BRCC36 IRIF in HT-29 cells 4 h post irradiation (10 Gy). Scale bar is 10 µm. e, Western blots showing BRCC36 protein levels in HT-29 cells reconstituted with WT or mutants BRCC36 as indicated (l eft ). Scatter plot showing quantification of the BRCC36 IRIF described in d . Data represent mean ± SEM derived from n ≥ 300 nuclei examined over two independent experiments; p values are indicated, unpaired two-tailed t test ( right ). f, K63-Ub2, -Ub4, and -Ub7 chains (1 µM) were incubated with ARISC WT or ARISC Δ42-55 (Abraxas1 Δ42-55) (5 nM) for up to 60 minutes. Cleavage activity was analysed as in a . Data are representative of two independent experiments. DUB, deubiquitylating enzyme; WT, wild type; Ub, ubiquitin.

Article Snippet: Reactions were stopped with the addition of 3 μL 4x SDS-PAGE loading dye [240 mM Tris-HCl pH 6.8, 40% (v/v) glycerol, 8% (w/v) SDS, 0.04% (w/v) bromophenol blue, and 5% (v/v) β-Mercaptoethanol], and products were separated on 4-12% or 12% Nu-PAGE Bis-Tris gels (Invitrogen).

Techniques: Incubation, Activity Assay, SDS Page, Silver Staining, Binding Assay, Mutagenesis, Irradiation, Western Blot, Derivative Assay, Two Tailed Test, Ubiquitin Proteomics