Vegfa

Manufactured by Sino Biological

Sourced in China

VEGFA is a growth factor that stimulates the formation of blood vessels. It is a key regulator of angiogenesis and plays a crucial role in various physiological and pathological processes.

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5 protocols using vegfa

Phage Display Screening for Antigen Binders

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BCscFv library was constructed (Fig. S1, Table S2) and characterized (Fig. S2).
The antigens, ubiquitin (R&D Systems), hemoglobin (Sigma), VEGFA (Sino Biological), VEGFR1 (R&D Systems), and CD44 (R&D Systems), were incubated in 96-well Nunc Maxisorp plates (Thermo Scientific) at 4 °C for 16–20 h for immobilization. For stringent phage selections, the amount of antigen for immobilization was gradually decreased: first round: 5 µg/mL, second round: 2.5 µg/mL, and third round: 1.25 μg/mL in PBS (100 µL). After each well was rinsed with PBST (PBS containing 0.05% Tween-20), non-specific binding sites were blocked with alternating proteins (300 μL) to prevent the emergence of phage that bind the blocking protein for 2 h (first round: 0.5% BSA in PBS, second round: 0.5% ovalbumin in PBS, and third round: 0.5% casein in PBS). The plate was rinsed with PBST, and phages in blocking buffer (100 μL, 10¹³ CFU/mL) were added to the plates for affinity selection and incubated for 2 h at room temperature. The wells were rinsed three times with PBST in the first and second rounds of screening and ten times in the third round of screening. The remaining scFv-expressing phages were eluted by a treatment with trypsin (100 μL/well, 1 mg/mL in PBS) for 10 min. The eluted phage were collected and stored at 4 °C for titration or amplification.

Wang X., Kim H.Y., Wahlberg B, & Edwards W.B. (2015). Selection and characterization of high affinity VEGFR1 antibodies from a novel human binary code scFv phage library. Biochemistry and Biophysics Reports, 3, 169-174.

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Cell Culture Conditions Across Cell Lines

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A549 (human lung adenocarcinoma), HEK293 (human embryonic kidney cells), HEK293A, HEK293T were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma D6429) containing 10% FBS, and 1% P/S (Invitrogen). For MDA-MB231 (invasive ductal breast carcinoma), DMEM was supplemented with 10% FBS, 1% P/S, and 1% non-essential amino acids (Sigma M7145). MCF10A (non-tumorigenic mammary epithelial cell line) were maintained in DMEM/F12 medium (Sigma D6421) containing 5% Horse Serum (Sigma H1270), 0.25 mM l-glutamine (Sigma G7513), 10 μg ml⁻¹ Insulin (Sigma I6634), 20 ng ml⁻¹ hEGF (Sigma E4269), 100 ng mL⁻¹ Cholera toxin (Sigma C8052), 0.5 μg ml⁻¹ Hydrocortisone (Sigma H4001), and 1% P/S. All cells were kept at 37 °C with 5% CO₂. BOEC, Telo-HEC, and HUVEC (Lonza CC-2519 pooled donor) were cultured in cEGM-2 media (Lonza). LATS1/2 and MST1/2 knockout HEK293A cells were established as described^{30 (link)} and cultured in DMEM/10% FBS/1% P/S. For some experiments, cells were stimulated with 100 ng ml⁻¹ VEGF-A (Sino Biological Inc.) after being serum-starved for 16 h.

Azad T., Janse van Rensburg H.J., Lightbody E.D., Neveu B., Champagne A., Ghaffari A., Kay V.R., Hao Y., Shen H., Yeung B., Croy B.A., Guan K.L., Pouliot F., Zhang J., Nicol C.J, & Yang X. (2018). A LATS biosensor screen identifies VEGFR as a regulator of the Hippo pathway in angiogenesis. Nature Communications, 9, 1061.

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Phage Display Library Construction and Screening

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BCscFv library was constructed (Figure S1, Table S2) and characterized (Figure S2).
The antigens, ubiquitin (R&D Systems), hemoglobin (Sigma), VEGFA (Sino Biological), VEGFR1 (R&D Systems), and CD44 (R&D Systems), were incubated in 96-well Nunc Maxisorp plates (Thermo Scientific) at 4 °C for 16 to 20 h for immobilization. For stringent phage selections, the amount of antigen for immobilization was gradually decreased: first round: 5 µg/mL, second round: 2.5 µg/mL, and third round: 1.25 µg/mL in PBS (100 µL). After each well was rinsed with PBST (PBS containing 0.05% Tween-20), non-specific binding sites were blocked with alternating proteins (300 µL) to prevent the emergence of phage that bind the blocking protein for 2 h (first round: 0.5% BSA in PBS, second round: 0.5% ovalbumin in PBS, and third round: 0.5% casein in PBS). The plate was rinsed with PBST, and phages in blocking buffer (100 µL, 10¹³ CFU/mL) were added to the plates for affinity selection and incubated for 2 h at room temperature. The wells were rinsed three times with PBST in the first and second rounds of screening and ten times in the third round of screening. The remaining scFv-expressing phages were eluted by a treatment with trypsin (100 µL/well, 1 mg/mL in PBS) for 10 min. The eluted phage were collected and stored at 4 °C for titration or amplification.

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Directed Differentiation of hESCs and hiPSCs

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H1, H9 hESCs (WiCell Institute), MESP1‐mTomato reporter hESC line,^[³¹ (link)
^] H2B‐mCherry hESC line,^[⁷⁰
^] and hiPSCs^[⁷¹ (link)
^] were cultured on Matrigel (BD Biosciences)‐coated plates (Corning) in TeSR‐E8 medium (STEMCELL Technologies). The chemically defined medium AATS for differentiation was described in the previous study.^[³⁰ (link)
^] For mesoderm progenitor cell induction, 5 ng mL⁻¹ BMP4 (Peprotech) and 2 µm mL⁻¹ CHIR99021 (Tocris) were added at the desired concentration, depending on the cell line. For EC induction, 50 ng mL⁻¹ VEGF‐A (SinoBiological) and 10 ng mL⁻¹ bFGF (FGF2, SinoBiological) were added. ROCK inhibitor Y27632 (TOCRIS) (5 µm) was added during passaging for 24 h and removed with the medium change.

Li M., Wang P., Huo S.T., Qiu H., Li C., Lin S., Guo L., Ji Y., Zhu Y., Liu J., Guo J., Na J, & Hu Y. (2023). Human Pluripotent Stem Cells Derived Endothelial Cells Repair Choroidal Ischemia. Advanced Science, 11(9), 2302940.

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Modulation of miR-520a in CRC

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MiR-520a mimics and inhibitor were synthesized by GenePharma (Shanghai, China) and transfected into HCT116 and SW480 cells by Lipofectamine 2000 (Invitrogen).
VEGFA was obtained from Sino Biological Inc. (Beijing, China) and dissolved in sterile water. 20 ng/ml VEGFA was mixed with CM from HCT116 with ATAD2 knockdown to culture HUVEC.

Hong S., Chen S., Wang X., Sun D., Yan Z., Tai J, & Bi M. (2018). ATAD2 silencing decreases VEGFA secretion through targeting has-miR-520a to inhibit angiogenesis in colorectal cancer. Biochemistry and cell biology = Biochimie et biologie cellulaire, 96(6).

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