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loading buffer  (Thermo Fisher)


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    Thermo Fisher loading buffer
    Loading Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/laemmli+loading+buffer/Laemmli+SDS+sample+buffer%2C+reducing/pmc13315006-219-8-10
    Average 99 stars, based on 1 article reviews
    loading buffer - by Bioz Stars, 2026-10
    99/100 stars

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    Related Articles

    Bicinchoninic Acid Protein Assay:

    Article Title: Bispecific antibody-drug conjugates targeting EGFR and LGR5 exert potent antitumor activity in colorectal cancer models
    Article Snippet: .. Protein concentrations were quantified by BCA protein assay (Thermo Scientific) and lysates were diluted in reducing SDS Laemmli buffer (Thermo Scientific) and incubated at 37°C for 1 h prior to SDS-PAGE. .. Anti-mouse IgG and anti-rabbit IgG HRP-labeled secondary antibodies from Cell Signaling Technology were utilized with the standard ECL protocol (Cytiva).

    Article Title: Neurodegenerative NMNAT2 Deficiency Promotes APP Processing in a SARM1-Dependent Manner.
    Article Snippet: Neurons were lysed using 150 μL of RIPA buffer per well [20 mM of Tris-base, 150 mM of NaCl, 2 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1× cOmpleteTM Mini protease inhibitor cocktail (Roche®, Basel, Switzerland), and phosphatase inhibitor cocktail 3 (Sigma-Aldrich® P0044, St. Louis, MO, USA), pH 7.4] and homogenized for 15 s with a pestle motor using disposable pellet pestles. .. Samples were then sonicated using a Branson Sonifier® 250 (Emerson, St. Louis, MO, USA) for 10 intervals set at duty cycle 30/output 3 and stored at −80 ◦C. https://doi.org/10.3390/cells15121100 Protein concentration was measured using the PierceTM BCA Protein Assay Kit (Thermo ScientificTM, Waltham, MA, USA) and samples were prepared for loading in Laemmli SDS buffer (Thermo ScientificTM). ..

    Incubation:

    Article Title: Bispecific antibody-drug conjugates targeting EGFR and LGR5 exert potent antitumor activity in colorectal cancer models
    Article Snippet: .. Protein concentrations were quantified by BCA protein assay (Thermo Scientific) and lysates were diluted in reducing SDS Laemmli buffer (Thermo Scientific) and incubated at 37°C for 1 h prior to SDS-PAGE. .. Anti-mouse IgG and anti-rabbit IgG HRP-labeled secondary antibodies from Cell Signaling Technology were utilized with the standard ECL protocol (Cytiva).

    Sonication:

    Article Title: Neurodegenerative NMNAT2 Deficiency Promotes APP Processing in a SARM1-Dependent Manner.
    Article Snippet: Neurons were lysed using 150 μL of RIPA buffer per well [20 mM of Tris-base, 150 mM of NaCl, 2 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1× cOmpleteTM Mini protease inhibitor cocktail (Roche®, Basel, Switzerland), and phosphatase inhibitor cocktail 3 (Sigma-Aldrich® P0044, St. Louis, MO, USA), pH 7.4] and homogenized for 15 s with a pestle motor using disposable pellet pestles. .. Samples were then sonicated using a Branson Sonifier® 250 (Emerson, St. Louis, MO, USA) for 10 intervals set at duty cycle 30/output 3 and stored at −80 ◦C. https://doi.org/10.3390/cells15121100 Protein concentration was measured using the PierceTM BCA Protein Assay Kit (Thermo ScientificTM, Waltham, MA, USA) and samples were prepared for loading in Laemmli SDS buffer (Thermo ScientificTM). ..

    Protein Concentration:

    Article Title: Neurodegenerative NMNAT2 Deficiency Promotes APP Processing in a SARM1-Dependent Manner.
    Article Snippet: Neurons were lysed using 150 μL of RIPA buffer per well [20 mM of Tris-base, 150 mM of NaCl, 2 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1× cOmpleteTM Mini protease inhibitor cocktail (Roche®, Basel, Switzerland), and phosphatase inhibitor cocktail 3 (Sigma-Aldrich® P0044, St. Louis, MO, USA), pH 7.4] and homogenized for 15 s with a pestle motor using disposable pellet pestles. .. Samples were then sonicated using a Branson Sonifier® 250 (Emerson, St. Louis, MO, USA) for 10 intervals set at duty cycle 30/output 3 and stored at −80 ◦C. https://doi.org/10.3390/cells15121100 Protein concentration was measured using the PierceTM BCA Protein Assay Kit (Thermo ScientificTM, Waltham, MA, USA) and samples were prepared for loading in Laemmli SDS buffer (Thermo ScientificTM). ..

    Western Blot:

    Article Title: MAPK/ERK signaling regulates H3K9me3 heterochromatin reorganization to confer mesendoderm developmental competence.
    Article Snippet: Protein concentration was measured using the Pierce icinchoninic acid (BCA) protein assay kit (23 225, Thermo isher Scientific). .. For western blot analysis, 5–15 μg of whole cell lysate in Laemmli Sample Buffer (J60015, Alfa Aesar) was denatured at 100◦C for 5 min, then separated on a 10%–15% sodium dodecyl sulphate–polyacrylamide gel electrophoresis gel at 60 V for 30 min, followed by 100 V for 2–2.5 h. Proteins on the gel were transferred to a 0.2 μm pore size nitrocellulose membrane (1620 112, Bio-Rad) using a wet tank transfer system with the Mini Blot Module (Thermo Fisher Scientific) at 10 V for 1 h. The transferred membrane was briefly washed with tween tris-buffered saline (TTBS) (0.05% Tween-20 in TBS) and blocked with blocking buffer [5% Blotting-Grade Blocker (1706 404, Bio-Rad) in TBS] for 1 h at room temperature. .. maging data processing enoising: Acquired images were denoised using the “Deoise.ai” function in NIS Elements Advanced Research (verion 5.20/5.30/5.42, Nikon Instrument).

    Nucleic Acid Electrophoresis:

    Article Title: MAPK/ERK signaling regulates H3K9me3 heterochromatin reorganization to confer mesendoderm developmental competence.
    Article Snippet: Protein concentration was measured using the Pierce icinchoninic acid (BCA) protein assay kit (23 225, Thermo isher Scientific). .. For western blot analysis, 5–15 μg of whole cell lysate in Laemmli Sample Buffer (J60015, Alfa Aesar) was denatured at 100◦C for 5 min, then separated on a 10%–15% sodium dodecyl sulphate–polyacrylamide gel electrophoresis gel at 60 V for 30 min, followed by 100 V for 2–2.5 h. Proteins on the gel were transferred to a 0.2 μm pore size nitrocellulose membrane (1620 112, Bio-Rad) using a wet tank transfer system with the Mini Blot Module (Thermo Fisher Scientific) at 10 V for 1 h. The transferred membrane was briefly washed with tween tris-buffered saline (TTBS) (0.05% Tween-20 in TBS) and blocked with blocking buffer [5% Blotting-Grade Blocker (1706 404, Bio-Rad) in TBS] for 1 h at room temperature. .. maging data processing enoising: Acquired images were denoised using the “Deoise.ai” function in NIS Elements Advanced Research (verion 5.20/5.30/5.42, Nikon Instrument).

    Pore Size:

    Article Title: MAPK/ERK signaling regulates H3K9me3 heterochromatin reorganization to confer mesendoderm developmental competence.
    Article Snippet: Protein concentration was measured using the Pierce icinchoninic acid (BCA) protein assay kit (23 225, Thermo isher Scientific). .. For western blot analysis, 5–15 μg of whole cell lysate in Laemmli Sample Buffer (J60015, Alfa Aesar) was denatured at 100◦C for 5 min, then separated on a 10%–15% sodium dodecyl sulphate–polyacrylamide gel electrophoresis gel at 60 V for 30 min, followed by 100 V for 2–2.5 h. Proteins on the gel were transferred to a 0.2 μm pore size nitrocellulose membrane (1620 112, Bio-Rad) using a wet tank transfer system with the Mini Blot Module (Thermo Fisher Scientific) at 10 V for 1 h. The transferred membrane was briefly washed with tween tris-buffered saline (TTBS) (0.05% Tween-20 in TBS) and blocked with blocking buffer [5% Blotting-Grade Blocker (1706 404, Bio-Rad) in TBS] for 1 h at room temperature. .. maging data processing enoising: Acquired images were denoised using the “Deoise.ai” function in NIS Elements Advanced Research (verion 5.20/5.30/5.42, Nikon Instrument).

    Membrane:

    Article Title: MAPK/ERK signaling regulates H3K9me3 heterochromatin reorganization to confer mesendoderm developmental competence.
    Article Snippet: Protein concentration was measured using the Pierce icinchoninic acid (BCA) protein assay kit (23 225, Thermo isher Scientific). .. For western blot analysis, 5–15 μg of whole cell lysate in Laemmli Sample Buffer (J60015, Alfa Aesar) was denatured at 100◦C for 5 min, then separated on a 10%–15% sodium dodecyl sulphate–polyacrylamide gel electrophoresis gel at 60 V for 30 min, followed by 100 V for 2–2.5 h. Proteins on the gel were transferred to a 0.2 μm pore size nitrocellulose membrane (1620 112, Bio-Rad) using a wet tank transfer system with the Mini Blot Module (Thermo Fisher Scientific) at 10 V for 1 h. The transferred membrane was briefly washed with tween tris-buffered saline (TTBS) (0.05% Tween-20 in TBS) and blocked with blocking buffer [5% Blotting-Grade Blocker (1706 404, Bio-Rad) in TBS] for 1 h at room temperature. .. maging data processing enoising: Acquired images were denoised using the “Deoise.ai” function in NIS Elements Advanced Research (verion 5.20/5.30/5.42, Nikon Instrument).

    Saline:

    Article Title: MAPK/ERK signaling regulates H3K9me3 heterochromatin reorganization to confer mesendoderm developmental competence.
    Article Snippet: Protein concentration was measured using the Pierce icinchoninic acid (BCA) protein assay kit (23 225, Thermo isher Scientific). .. For western blot analysis, 5–15 μg of whole cell lysate in Laemmli Sample Buffer (J60015, Alfa Aesar) was denatured at 100◦C for 5 min, then separated on a 10%–15% sodium dodecyl sulphate–polyacrylamide gel electrophoresis gel at 60 V for 30 min, followed by 100 V for 2–2.5 h. Proteins on the gel were transferred to a 0.2 μm pore size nitrocellulose membrane (1620 112, Bio-Rad) using a wet tank transfer system with the Mini Blot Module (Thermo Fisher Scientific) at 10 V for 1 h. The transferred membrane was briefly washed with tween tris-buffered saline (TTBS) (0.05% Tween-20 in TBS) and blocked with blocking buffer [5% Blotting-Grade Blocker (1706 404, Bio-Rad) in TBS] for 1 h at room temperature. .. maging data processing enoising: Acquired images were denoised using the “Deoise.ai” function in NIS Elements Advanced Research (verion 5.20/5.30/5.42, Nikon Instrument).

    Blocking Assay:

    Article Title: MAPK/ERK signaling regulates H3K9me3 heterochromatin reorganization to confer mesendoderm developmental competence.
    Article Snippet: Protein concentration was measured using the Pierce icinchoninic acid (BCA) protein assay kit (23 225, Thermo isher Scientific). .. For western blot analysis, 5–15 μg of whole cell lysate in Laemmli Sample Buffer (J60015, Alfa Aesar) was denatured at 100◦C for 5 min, then separated on a 10%–15% sodium dodecyl sulphate–polyacrylamide gel electrophoresis gel at 60 V for 30 min, followed by 100 V for 2–2.5 h. Proteins on the gel were transferred to a 0.2 μm pore size nitrocellulose membrane (1620 112, Bio-Rad) using a wet tank transfer system with the Mini Blot Module (Thermo Fisher Scientific) at 10 V for 1 h. The transferred membrane was briefly washed with tween tris-buffered saline (TTBS) (0.05% Tween-20 in TBS) and blocked with blocking buffer [5% Blotting-Grade Blocker (1706 404, Bio-Rad) in TBS] for 1 h at room temperature. .. maging data processing enoising: Acquired images were denoised using the “Deoise.ai” function in NIS Elements Advanced Research (verion 5.20/5.30/5.42, Nikon Instrument).

    SDS Page:

    Article Title: Physicochemical stability and protein corona profiling on the interaction of iron oxide nanoparticles with human tears.
    Article Snippet: .. SDS-PAGE of tear extract controls and unbound or loosely bound (soft corona) proteins and the IONP extracted proteins, 10 mL of each protein samples were mixed separately with 10 mL of 2 Laemmli buffer (Invitrogen) and heated at 95 1C for 5 minutes to ensure complete protein denaturation. .. The prepared samples, along with 2 mL of Benchmarkt pre-stained protein molecular weight ladder (Invitrogen), were loaded onto a 12% Tris-polyacrylamide gel and electrophoresed in Trisglycine running buffer (Invitrogen) at 120 V for 45 minutes.

    Protease Inhibitor:

    Article Title: Immortalization of human airway epithelial cells for genome editing applications
    Article Snippet: .. Differentiated cells were trypsinized and lysed in Laemmli buffer (120 mM Tris-HCl pH 6.8, 20 % glycerol, 4 % sodium dodecyl sulfate (SDS), protease inhibitor (Fisher Emergo B.V.)), and the lysates were homogenized using a QIAshredder (QIAGEN N.V., Germany) column to reduce viscosity. .. Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA) according to the manufacturer’s instructions.

    Viscosity:

    Article Title: Immortalization of human airway epithelial cells for genome editing applications
    Article Snippet: .. Differentiated cells were trypsinized and lysed in Laemmli buffer (120 mM Tris-HCl pH 6.8, 20 % glycerol, 4 % sodium dodecyl sulfate (SDS), protease inhibitor (Fisher Emergo B.V.)), and the lysates were homogenized using a QIAshredder (QIAGEN N.V., Germany) column to reduce viscosity. .. Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA) according to the manufacturer’s instructions.

    Isolation:

    Article Title: The club cell circadian clock regulates temporal patterns in leukocyte trafficking in chronic allergic airways disease
    Article Snippet: Mouse lungs were weighed and approximately 30–35 mg of tissue was lysed using 0.5 ml T-Per tissue protein extraction reagent (Thermo Scientific) supplemented with protease and phosphatase inhibitor cocktail (cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail, MilliporeSigma) using a lysing matrix D on a homogenizer. .. Proteins were isolated from the lysate supernatant and quantified using Pierce BCA assay, and 50 μg of protein was heated at 95 °C for 5 min in Laemmli SDS sample reducing buffer (Thermo Scientific) and loaded into Any kD Mini-PROTEAN TGX Precast Protein Gels (BioRad). .. Gels were run using premixed electrophoresis buffer (BioRad) at 100 V for an hour.

    BIA-KA:

    Article Title: The club cell circadian clock regulates temporal patterns in leukocyte trafficking in chronic allergic airways disease
    Article Snippet: Mouse lungs were weighed and approximately 30–35 mg of tissue was lysed using 0.5 ml T-Per tissue protein extraction reagent (Thermo Scientific) supplemented with protease and phosphatase inhibitor cocktail (cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail, MilliporeSigma) using a lysing matrix D on a homogenizer. .. Proteins were isolated from the lysate supernatant and quantified using Pierce BCA assay, and 50 μg of protein was heated at 95 °C for 5 min in Laemmli SDS sample reducing buffer (Thermo Scientific) and loaded into Any kD Mini-PROTEAN TGX Precast Protein Gels (BioRad). .. Gels were run using premixed electrophoresis buffer (BioRad) at 100 V for an hour.



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    2x laemmli loading buffer - by Bioz Stars, 2026-10
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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 , 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