sds page loading dye  (Bio-Rad)

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