How much of H00003091-T01 typically needs to be loaded on a gel - 10uL, 5uL?

In regards to your inquiry this is a lysate that we distribute for a Taiwanese company called Abnova. I pulled up my information and they recommend loading 25 uL.

I would like to know what dilution buffer is recommended?

The recommended dilution buffer is 25uL per well of PBS.

HIF-1 alpha 293T Cell Transient Overexpression Lysate

Name: HIF-1 alpha 293T Cell Transient Overexpression Lysate
Brand: Novus Biologicals
SKU: H00003091-T01

Novus Biologicals, part of Bio-Techne | Catalog # H00003091-T01

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Product Summary
Product Specifications
Scientific Data
Preparation & Storage
Background
Product Documents
Specific Notices

Key Product Details

Species

Human

Applications

Western Blot

Product Summary for HIF-1 alpha 293T Cell Transient Overexpression Lysate

Quality control test: Transient overexpression cell lysate was tested with Anti-HIF1A antibody by Western Blots. Plasmid: pCMV-HIF1A full-length

Product Specifications

Specificity

HIF-1 alpha 293T Cell Transient Overexpression Lysate(Denatured)

Type

293T Cell Transient Overexpression

Protein State

Denatured

Scientific Data Images for HIF-1 alpha 293T Cell Transient Overexpression Lysate

Lane 1: HIF1A transfected lysate (82.7 KDa) Lane 2: Non-transfected lysate.

Other: HIF-1 alpha 293T Cell Transient Overexpression Lysate [H00003091-T01]

HIF1A transfected lysate.

Formulation, Preparation, and Storage

Formulation

1X Sample Buffer (50 mM Tris-HCl, 2% SDS, 10% glycerol, 300 mM 2-mercaptoethanol, 0.01% Bromophenol blue)

Concentration

mg/ml

Shipping

The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.

Storage

Store at -80C. Avoid freeze-thaw cycles.

Background: HIF-1 alpha/HIF1A

Hypoxia contributes to the pathophysiology of human disease, including myocardial and cerebral ischemia, cancer, pulmonary hypertension, congenital heart disease and chronic obstructive pulmonary disease (1). In cancer and particularly solid tumors, hypoxia plays a critical role in the regulation of genes involved in stem cell renewal, epithelial to mesenchymal transition (EMT), metastasis and angiogenesis. In the tumor microenvironment (TME), hypoxia influences the properties and function of stromal cells (e.g., fibroblasts, endothelial and immune cells) and is a strong determinant of tumor progression (2,3).

HIF-1 or hypoxia inducible factor 1 (predicted molecular weight 93kDa), is a transcription factor commonly referred to as a "master regulator of the hypoxic response" for its central role in the regulation of cellular adaptations to hypoxia. In its active form under hypoxic conditions, HIF-1 is stabilized by the formation of a heterodimer of HIF-1 alpha and ARNT/HIF-1 beta subunits. Nuclear HIF-1 engages p300/CBP for binding to hypoxic response elements (HREs). This process induces transcription and regulation of genes including EPO, VEGF, iNOS2, ANGPT1 and OCT4 (4,5).

Under normoxic conditions, the HIF-1 alpha subunit is rapidly targeted and degraded by the ubiquitin proteasome system. This process is mediated by prolyl hydroxylase domain enzymes (PHDs), which catalyze the hydroxylation of key proline residues (Pro-402 and Pro-564) within the oxygen-dependent degradation domain of HIF-1 alpha. Once hydroxylated, HIF-1 alpha binds the von Hippel-Lindau tumor suppressor protein (pVHL) for subsequent ubiquitination and proteasomal degradation (4). pVHL dependent regulation of HIF-1 alpha plays a role in normal physiology and disease states. Regulation of HIF-1 alpha by pVHL is critical for the suppressive function of FoxP3+ regulatory Tcells (6). Repression of pVHL expression in chronic lymphocytic leukemia (CLL) B cells leads to HIF-1 alpha stabilization and increased VEGF secretion (7).

References

1. Semenza, G. L., Agani, F., Feldser, D., Iyer, N., Kotch, L., Laughner, E., & Yu, A. (2000). Hypoxia, HIF-1, and the pathophysiology of common human diseases. Advances in Experimental Medicine and Biology.

2. Muz, B., de la Puente, P., Azab, F., & Azab, A. K. (2015). The role of hypoxia in cancer progression, angiogenesis, metastasis, and resistance to therapy. Hypoxia. https://doi.org/10.2147/hp.s93413

3. Huang, Y., Lin, D., & Taniguchi, C. M. (2017). Hypoxia inducible factor (HIF) in the tumor microenvironment: friend or foe? Science China Life Sciences. https://doi.org/10.1007/s11427-017-9178-y

4. Koyasu, S., Kobayashi, M., Goto, Y., Hiraoka, M., & Harada, H. (2018). Regulatory mechanisms of hypoxia-inducible factor 1 activity: Two decades of knowledge. Cancer Science. https://doi.org/10.1111/cas.13483

5. Dengler, V. L., Galbraith, M. D., & Espinosa, J. M. (2014). Transcriptional regulation by hypoxia inducible factors. Critical Reviews in Biochemistry and Molecular Biology. https://doi.org/10.3109/10409238.2013.838205

6. Lee, J. H., Elly, C., Park, Y., & Liu, Y. C. (2015). E3Ubiquitin Ligase VHL Regulates Hypoxia-Inducible Factor-1 alpha to Maintain Regulatory T Cell Stability and Suppressive Capacity. Immunity. https://doi.org/10.1016/j.immuni.2015.05.016

7. Ghosh, A. K., Shanafelt, T. D., Cimmino, A., Taccioli, C., Volinia, S., Liu, C. G., ... Kay, N. E. (2009). Aberrant regulation of pVHL levels by microRNA promotes the HIF/VEGF axis in CLL B cells. Blood. https://doi.org/10.1182/blood-2008-10-185686

Long Name

Hypoxia Inducible Factor 1 Subunit Alpha

Alternate Names

BHLHE78, HIF 1A, HIF-1a, HIF1 alpha, HIF1A, MOP1, PASD8

Gene Symbol

HIF1A

Additional HIF-1 alpha/HIF1A Products

Product Documents for HIF-1 alpha 293T Cell Transient Overexpression Lysate

Product Datasheets

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COA

Product Specific Notices for HIF-1 alpha 293T Cell Transient Overexpression Lysate

This product is produced by and distributed for Abnova, a company based in Taiwan.

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Lysates are guaranteed for 6 months from date of receipt.

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