sds page loading dye  (Bio-Rad)

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

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

Journal: bioRxiv

Article Title: Sugar Import Suppresses Klebsiella pneumoniae Mucoidy in cAMP-CRP-dependent Manner

doi: 10.1101/2025.11.04.686645

Figure Lengend Snippet: (A) KPPR1 carrying a GFP reporter vector (P rmp - dasherGFP ) or (B-D) unmodified KPPR1 were cultured in M9+CAA±Gal. (A) KPPR1 rmpADC promoter (P rmp ) activity was quantified by measuring the P rmp -driven GFP fluorescence intensity and normalized to OD 600 . (B) Relative transcript levels of KPPR1 rmpA , rmpD and rmpC were quantified by qRT-PCR in M9+CAA+Gal and compared to M9+CAA. (C) Cell-associated CPS was purified and resolved using a gradient SDS-PAGE and stained with 1% Alcian blue followed by silver stain. The presented gel image is representative of three independent experiments. (D) CPS chain length diversity was quantified by densitometric analysis using ImageJ. Data presented (A, B and D) are the mean, and the error bars represent the standard error of the mean. Statistical significance was determined using (A) unpaired t-test and (B and D) multiple unpaired t-tests with multiple-comparison correction using the Benjamini, Krieger and Yekutieli false discovery rate method. Statistical significance was calculated by comparing sugar-supplemented condition to sugar-deficient condition. ** p ≤ 0.01; # p ≤ 0.0001. Experiments were performed ≥3 independent times, in triplicate.

Article Snippet: Purified cell-associated CPS was mixed with 4 × SDS gel loading dye (3:1, sample:dye) and resolved using 4-15% TGX stain-free precast gel (Bio-Rad).

Techniques: Plasmid Preparation, Cell Culture, Activity Assay, Fluorescence, Quantitative RT-PCR, Purification, SDS Page, Staining, Silver Staining, Comparison

Journal: bioRxiv

Article Title: Sugar Import Suppresses Klebsiella pneumoniae Mucoidy in cAMP-CRP-dependent Manner

doi: 10.1101/2025.11.04.686645

Figure Lengend Snippet: KPPR1 wildtype (WT), and cyaA and crp mutants were cultured in M9+CAA±Gal. (A) Mucoidy was determined by quantifying the supernatant OD 600 after centrifugation at 1,000 x g for 5 mins. (B) Cell-associated CPS was extracted and measured for uronic acid content. (C) Intracellular cAMP levels and (D) P rmp activity were measured using a P rmp and cAMP-CRP-responsive dual fluorescent reporter. Cell-associated CPS were (E) resolved by SDS-PAGE and (F) analyzed for chain length diversity by densitometric analysis in ImageJ. Data presented are the mean, and the error bars represent the standard error of the mean. Statistical significance was determined using two-way ANOVA with Dunnett’s post-hoc test where each mutant was compared to WT in the same growth medium. * p ≤ 0.05; # p ≤ 0.0001. CPS staining was performed independently three times with a cumulative total of n = 3 biological replicates. All other experiments were performed ≥3 independent times, in triplicate.

Article Snippet: Purified cell-associated CPS was mixed with 4 × SDS gel loading dye (3:1, sample:dye) and resolved using 4-15% TGX stain-free precast gel (Bio-Rad).

Techniques: Cell Culture, Centrifugation, Activity Assay, SDS Page, Mutagenesis, Staining