streptavidin, horseradish peroxidase, concentrate, for ihc  (Vector Laboratories)

Journal: iScience

Article Title: Enzymatically controlled release of proteins and peptides: A promising, alternative secretion approach

doi: 10.1016/j.isci.2026.115185

Figure Lengend Snippet: Analysis of de novo RBD expression, secretion, and intracellular TEVp-mediated cleavage in co-transfected HEK293T cells (A) Overview of the experimental set-up to analyze the total RBD-V5-His release and TEVp- c -Myc-His secretion (left side) and de novo RBD-V5-His release (right side) from co-transfected HEK293T cells. (B) Western blot analysis of the samples derived from experiment (A) by detection of V5 or c -Myc tag. (C) Western blot analysis of the secreted and residual intracellular RBD-V5-His (top) and TEVp N23Q,C130S,T173G,S219V - c -Myc-His (bottom) isolated using His-tag from 1 mL of supernatant (left lanes) or lysed co-transfected HEK293T cells from one well of a 6-well plate in 1 mL (right lanes). Cells were lysed with 1% of IGEPAL CA-630, which was also added to the supernatant. (D) Coomassie Brilliant blue-stained polyacrylamide gel quantified by comparison with a bovine serum albumin standard curve (left) and subsequent western blot probed by anti-V5 antibodies (right) of the protein released from the supernatant of co-transfected HEK293T cells obtained by co-expression of tANCHORed RBD-V5-His (top) or CD63LEL-V5-His (bottom) with or without TEVp N23Q,C130S,T173G,S219V - c -Myc-His. Highly glycosylated CD63LEL-V5-His was treated with PNGase F (peptide N-glycosidase F). (E) Lectin blot of the released RBD-V5-His protein, probed with biotinylated Sambucus nigra agglutinin (SNA) and detected using streptavidin-HRP. (F and G) Western blot analysis of the cell lysates for the expression of CD63-mCherry (F) or CD82-tANCHOR-CD63LEL-mCherry (G), with or without modified TEVp expression. Control contains untransfected cells, and TEVp was captured from the supernatant.

Article Snippet: The membrane was then washed three times with 1× PBS and incubated with streptavidin-HRP (R&D Systems, Bio-Techne, Wiesbaden, Germany) in a dilution of 1:200 in 3% BSA for 45 min. Lectin-bound protein was detected using ECL substrate Atto SuperSignal substrate (Thermo Fisher Scientific).

Techniques: Expressing, Transfection, Western Blot, Derivative Assay, Isolation, Staining, Comparison, Modification, Control

Journal: The Journal of Cell Biology

Article Title: Landscape expansion microscopy reveals interactions between membrane and phase-separated organelles

doi: 10.1083/jcb.202502035

Figure Lengend Snippet: land-ExM visualizes phase-separated and membrane organelles. (A–G) land-ExM protein images of membraneless phase separation structures. The proteins were labeled with NHS-biotin-MS and after gelation stained with streptavidin-Alexa Fluor 488. (A) land-ExM protein image of nucleoli in a U2OS cell. Red arrowheads indicate the fibrillar center (FC) or dense fibrillar component (DFC) of the nucleolus. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.0. (B) land-ExM protein image of nuclear bodies of breast cancer cell, UCI082014. Red arrowheads indicate the nuclear bodies. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.2. (C) land-ExM protein image of SGs of a U2OS cell treated with NaAsO 2 for 20 min. The red arrowhead indicates a SG. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.0. (D) land-ExM protein image of chromatin of a breast cancer cell. Scale bar: 500 nm in pre-expansion unit. Linear expansion factor: 4.2. (E) land-ExM protein image of NPCs of a breast cancer cell. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.2. (F and G) land-ExM protein images of mitochondria and cytoskeleton of a U2OS cell. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.0. (H–P) land-ExM lipid images of membrane structures. The lipids were labeled with mCLING-Atto647N. (H) land-ExM lipid image of breast cancer cell. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4.0. (I–M) magnified images of H showing different membrane structures: lipid vesicles (I), mitochondria (J), filopodia (K), nuclear invagination (L), and Golgi apparatus (M). Scale bar: 1 µm (I–M) in pre-expansion unit. (N) 3D land-ExM lipid image of a breast cancer cell after maximum intensity projection, showing the cell membrane. Color-coded by the z-dimension slices from bottom to top. Color bar: purple to white: 0–6 µm in pre-expansion unit. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4.0. (O and P) magnified images of N showing detailed structures of the cell membrane. Scale bar: 1 µm in pre-expansion unit. All images were taken with an Airyscan microscope.

Article Snippet: The cell-gelation solution was then incubated at 37°C for 1–2 h. Gelled cells were immersed in heat denaturation buffer (200 mM sodium dodecyl sulfate, 200 mM NaCl, and 50 mM Tris, pH 6.8) for 1.5 h at 78°C and washed with excess of water for 30 min. To fluorescently label the whole proteins, immerse the gelled cells in poststaining buffer (10 mM HEPES and 150 mM NaCl, pH 7.5) twice, 30 min each time, then incubate the gelled cells with 4 μg/ml streptavidin dyes (included in cat#LD-P568-050/LD-P488-050; Cytoseen, or cat#016-540-084; Jackson ImmunoResearch) in poststaining buffer overnight.

Techniques: Membrane, Labeling, Staining, Microscopy

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Fas-induced T cell activation occurs in the absence of FADD. (A) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fadd -targeting gRNAs and polarized under Th2 conditions in the presence or absence of FasL-LZ (25 ng/ml). Top: Representative flow cytometry histograms showing pS6(S240/44) staining in live CD4 + cells. Bottom: Fold induction of pS6(S240/44) in Th2-polarized (−/+ FasL-LZ) live CD4 + T cells. Fold induction was calculated by normalizing MFIs to NC WT cells. n = 4–5 for each group, from four to five independent experiments. (B and C) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice were nucleofected with Cas9-gRNA complexes containing NC or Fadd -targeting gRNAs and polarized under Th2 conditions. n = 6 for each group, from six independent experiments. (B) Frequencies of IFNγ + Th2-polarized cells. (C) Th2-polarized cells were gated as FasL low , FasL mid , and FasL high . Frequencies of IFNγ + cells were measured in the indicated groups. (D–G) Fas was tagged with BioID2 on its intracellular C terminus (Fas-BioID) and stably expressed in Fas-deficient Jurkat cells. Fas-BioID Jurkat cells were cultured in the presence or absence of recombinant multimeric FasL (FasL-LZ). Jurkat cells expressing BioID alone were used as a control. (D) Venn diagram showing proteins identified following mass spectrometry analysis of biotinylated proteins (streptavidin pull-down) that were common between or specific to BioID-alone Jurkat cells versus Fas-BioID + FasL-LZ (P < 0.05, n = 3 for all groups). (E) Heatmap (row z-score) showing % normalized spectral abundance of proteins specifically upregulated in Fas-BioID ± FasL-LZ Jurkat cells relative to BioID-alone Jurkat cells. (F) Pathway enrichment of Reactome gene sets was performed using Enrichr , with significantly enriched gene sets colored in blue; proteins specifically upregulated in Fas-BioID + FasL-LZ Jurkat cells relative to BioID-alone Jurkat cells were used as input for pathway enrichment. (G) Normalized spectral abundance (%) of the indicated proteins in BioID-alone, Fas-BioID, and Fas-BioID + FasL-LZ Jurkat cells, measured by mass spectrometry. Statistical comparisons were made using ratio paired t tests (G). *P < 0.05, **P < 0.01. NC, negative control.

Article Snippet: Biotinylated proteins were pulled down using streptavidin agarose beads (Pierce) at 4°C overnight.

Techniques: Activation Assay, Flow Cytometry, Staining, Stable Transfection, Cell Culture, Recombinant, Expressing, Control, Mass Spectrometry, Negative Control

Journal: The Journal of Cell Biology

Article Title: Landscape expansion microscopy reveals interactions between membrane and phase-separated organelles

doi: 10.1083/jcb.202502035

Figure Lengend Snippet: land-ExM visualizes the protein and lipid context of cells. (A) Workflow of land-ExM. (B) Schematic of NHS-biotin-MA linker. (C) Schematic of mCLING. (D) land-ExM image of U2OS cells incubated with NHS-biotin-MA linker. Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4. (E) ExM image of U2OS cells incubated with NHS-MA linker and stained with Alexa Fluor 488 NHS ester dye. Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.2. (F) ExM image of U2OS cells incubated with GMA linker and stained with SYPRO Orange. Scale bar: 10 µm in pre-expansion unit. Linear expansion factor: 4.2. (G) Bar chart comparing signal-to-noise ratios of protein context images obtained with different ExM methods shown in D–F. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 10 cells. (H–J) Different views of land-ExM images of a breast cancer cell, UCI082014, stained with mCLING for lipid content. The orange dashed lines in H show where the orthogonal views (I and J) align. Scale bar: 5 µm (H), 2 µm (I and J) in pre-expansion unit. Linear expansion factor: 3.8. (K) Magnified images of H. (L) Magnified images of I. The orange dashed line in K shows where the orthogonal view (L) aligns. Scale bar: 0.5 µm in pre-expansion unit. Linear expansion factor: 3.8. All images were taken with an Airyscan microscope. Images D–F were adjusted to the same contrast. Image in D is also shown in .

Article Snippet: Alexa Fluor 488 Streptavidin , Jackson ImmunoResearch , 016-540-084.

Techniques: Incubation, Staining, Microscopy

Journal: The Journal of Cell Biology

Article Title: Landscape expansion microscopy reveals interactions between membrane and phase-separated organelles

doi: 10.1083/jcb.202502035

Figure Lengend Snippet: land-ExM labeling and anchoring strategies improve the signal of TREx and pan-ExM. (A) Workflow of land-pan-ExM, which only replaces the labeling strategy of pan-ExM with the labeling strategy of land-ExM. (B) land-TREx protein channel of U2OS cells, where proteins were labeled and anchored with NHS-biotin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 7. (C) TREx protein channel of U2OS cells, where proteins were anchored with acryloyl-X SE and stained with Alexa Fluor 488 NHS ester. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 7. (D) Bar chart comparing the signal-to-noise ratio of the protein channel in land-TREx and TREx. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 20 cells. (E) land-TREx lipid channel of U2OS cells, where lipids were labeled by mCLING and anchored with NHS-biotin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 7.0. (F) TREx lipid channel of U2OS cells, where lipids were anchored with acryloyl-X SE and stained with mCLING. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 7.0. (G) Bar chart comparing the signal-to-noise ratio of the lipid channel of land-TREx and TREx. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 20 cells. (H) land-pan-ExM protein channel of U2OS cells, where proteins were labeled and anchored with NHS-biotin-MA. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 12.0. (I) Pan-ExM protein channel of U2OS cells labeled with Alexa Fluor 488 NHS ester. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 12.0. (J) Bar chart comparing the signal-to-noise ratio of the protein channel in land-pan-ExM and pan-ExM. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 20 cells. (K) land-pan-ExM lipid channel of U2OS cells, where lipids were stained following the workflow (A). Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 12.0. (L) Pan-ExM lipid channel of U2OS cells labeled with mCLING. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 12.0. (M) Bar chart comparing the signal-to-noise ratio of the lipid (mCLING) channel in land-pan-ExM and pan-ExM. The signal-to-noise ratio is calculated as the average pixel value of the area with cells divided by the average pixel value of the area without cells in each image. Each bar represents the mean ± standard error of more than 20 cells. All images were taken with an Airyscan microscope.

Article Snippet: Alexa Fluor 488 Streptavidin , Jackson ImmunoResearch , 016-540-084.

Techniques: Labeling, Staining, Microscopy

Journal: The Journal of Cell Biology

Article Title: Landscape expansion microscopy reveals interactions between membrane and phase-separated organelles

doi: 10.1083/jcb.202502035

Figure Lengend Snippet: land-ExM visualizes phase-separated and membrane organelles. (A–G) land-ExM protein images of membraneless phase separation structures. The proteins were labeled with NHS-biotin-MS and after gelation stained with streptavidin-Alexa Fluor 488. (A) land-ExM protein image of nucleoli in a U2OS cell. Red arrowheads indicate the fibrillar center (FC) or dense fibrillar component (DFC) of the nucleolus. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.0. (B) land-ExM protein image of nuclear bodies of breast cancer cell, UCI082014. Red arrowheads indicate the nuclear bodies. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.2. (C) land-ExM protein image of SGs of a U2OS cell treated with NaAsO 2 for 20 min. The red arrowhead indicates a SG. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.0. (D) land-ExM protein image of chromatin of a breast cancer cell. Scale bar: 500 nm in pre-expansion unit. Linear expansion factor: 4.2. (E) land-ExM protein image of NPCs of a breast cancer cell. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.2. (F and G) land-ExM protein images of mitochondria and cytoskeleton of a U2OS cell. Scale bar: 1 µm in pre-expansion unit. Linear expansion factor: 4.0. (H–P) land-ExM lipid images of membrane structures. The lipids were labeled with mCLING-Atto647N. (H) land-ExM lipid image of breast cancer cell. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4.0. (I–M) magnified images of H showing different membrane structures: lipid vesicles (I), mitochondria (J), filopodia (K), nuclear invagination (L), and Golgi apparatus (M). Scale bar: 1 µm (I–M) in pre-expansion unit. (N) 3D land-ExM lipid image of a breast cancer cell after maximum intensity projection, showing the cell membrane. Color-coded by the z-dimension slices from bottom to top. Color bar: purple to white: 0–6 µm in pre-expansion unit. Scale bar: 5 µm in pre-expansion unit. Linear expansion factor: 4.0. (O and P) magnified images of N showing detailed structures of the cell membrane. Scale bar: 1 µm in pre-expansion unit. All images were taken with an Airyscan microscope.

Article Snippet: Alexa Fluor 488 Streptavidin , Jackson ImmunoResearch , 016-540-084.

Techniques: Membrane, Labeling, Staining, Microscopy