Recombinant TGFB1 1mg
Expression system/Source: Protein, purified, expressed in CHO, animal‐free product.
Target formulation: Lyophilized from a 0.2 μM filtered solution in 30 mM Sodium Citrate, 50 mM NaCl, 10nM Glycine pH 4.0
Purity: >95%, by SDS-PAGE with Coomassie staining and HPLC
Endotoxin Level: <0.01 EU per 1 μg of the protein by the LAL method.
UniProt Accession #: P01137
Structure / Isoform: Dimer
Predicted Molecular Mass/ Molecular weight: 12793.58 Da, determined by high-resolution TOF-MS to confirm correct protein sequence.
~13 kDa, reducing condition, ~25 kDa, non-reducing conditions.
UniProt Description of TGF:
Multifunctional protein that regulates the growth and differentiation of various cell types and is involved in various processes, such as normal development, immune function, microglia function and responses to neurodegeneration (By similarity). Activation into mature form follows different steps: following cleavage of the proprotein in the Golgi apparatus, Latency-associated peptide (LAP) and Transforming growth factor beta-1 (TGF-beta-1) chains remain non-covalently linked rendering TGF-beta-1 inactive during storage in extracellular matrix (PubMed:29109152). At the same time, LAP chain interacts with 'milieu molecules', such as LTBP1, LRRC32/GARP and LRRC33/NRROS that control activation of TGF-beta-1 and maintain it in a latent state during storage in extracellular milieus (PubMed:2022183, PubMed:8617200, PubMed:8939931, PubMed:19750484, PubMed:22278742, PubMed:19651619). TGF-beta-1 is released from LAP by integrins (ITGAV: ITGB6 or ITGAV: ITGB8): integrin-binding to LAP stabilizes an alternative conformation of the LAP bowtie tail and results in distortion of the LAP chain and subsequent release of the active TGF-beta-1 (PubMed:22278742, PubMed:28117447). Once activated following release of LAP, TGF-beta-1 acts by binding to TGF-beta receptors (TGFBR1 and TGFBR2), which transduce signal (PubMed:20207738).
ALDTNYCFSS TEKNCCVRQL YIDFRKDLGW KWIHEPKGYH ANFCLGPCPY IWSLDTQYSK VLALYNQHNP GASAAPCCVP QALEPLPIVY YVGRKPKVEQ LSNMIVRSCKCS
BIOACTIVITY ASSAY SUMMARY PROTOCOL
The bioactivity (ED50) of TGFb1 is determined in a 48-hour inhibition of proliferation assay using TF-1 erythroleukemia suspension cells (ATCC CRL-2003) in the presence of 2 ng/mL granulocyte-macrophage colony stimulating factor (GM-CSF), serum free media and CellTiter-Glo® Luminescent Cell Viability Assay (Promega G7570).
1. A stock solution of TGFb1 is prepared with sterile DPBS and the concentration is confirmed using a NanoDrop spectrophotometer or equivalent piece of equipment.
2. TF-1 suspension cells are seeded at 10,000 cells per well in a 96 well plate in serum free DMEM media (DMEM with 0.1% Bovine Serum Albumin) on Day 0.
3. A titration of TGFb1 is prepared in serum free DMEM at 2X concentration by serial dilution and added at equal volume to the cell suspension and incubated for 48 hours in a 37⁰C 5% CO2 incubator.
4. On Day 2, CellTiter-Glo® is added to the plate and luminescence is measured on a plate reader.
5. ED50 analysis is performed and recorded.
Full-Length Protocol Can Be Found Here: PDF
PREPARATION AND STORAGE
1. Reconstitute at 1 mg/mL in distilled water (final buffer composition 30 mM Sodium citrate, 50 mM NaCl, 10mM Glycine, pH 4.0)
a. It is recommended to filter the solution after reconstitution.
2. The product is shipped with cold packs. Upon receipt, store it immediately at the temperature recommended below.
3. Avoid repeated freeze‐thaw cycles. A minimum of 6 months when stored at ≤ ‐20 °C as supplied. Refer to lot specific COA for the Use by Date. 1 month, 2 to 4 °C under sterile conditions after reconstitution. 3 months, ≤ ‐20 °C under sterile conditions after reconstitution.
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