Qk005 recombinant human Activin A Plus 2.1 (ActA+ 2.1) – optimised
Protein quality information for Qk005 batch #010
All our proteins are produced in-house by our scientists and we provide detailed quality data for each individual batch. Please contact us any time by email firstname.lastname@example.org or phone +44 (0) 1223 491486 if you have any questions.
After aliquotting and lyophilising the protein, we choose a vial at random and reconstitute as recommended to ensure we are testing as close to the protein you will receive as possible. Biochemical identity and purity of each batch is checked using SDS-PAGE, mass spectrometry and analytical reverse phase chromatography. We use a sensitive test to ensure endotoxin levels are at industry leading low levels (<0.01 EU per µg protein). Bioactivity of the protein is determined using the quantitative activin responsive luciferase reporter assay. We also check that the correct amount of protein is recovered from the vial – it might sound basic but if you order 100 µg, we believe you should receive 100 µg.
Result: ActA+ 2.1 migrates as a single band at 24 kDa in non-reducing (NR) and 13 kDa as a single monomeric species upon reduction (R). No contaminating protein bands are visible.
Purified recombinant protein (7 µg) was resolved using 15% w/v SDS-PAGE in reduced (+β-mercaptothanol, R) and non-reduced conditions (NR) and stained with Coomassie Brilliant Blue R250.
Result: ActA 2.1 activity is determined using an activin responsive firefly luciferase reporter in HEK293T cells. EC50 = 5.6 pM . EC50 is within the expected range of 6 ± 2 pM.
Bioactivity is determined using an activin responsive firefly luciferase reporter in HEK293T cells. Cells are treated (in triplicate) with a serial dilution of activin A for 6 hours. Firefly luciferase activity is measured and normalised to the control Renilla luciferase activity.
Result: calculated molecular mass of the Activin A dimer is 24365.82 Da. Result of the analysis:24382.0 Da which is consistent with the calculated mass. No significant heterogeneity is present.
MALDI mass spectrometric analysis is used to confirm the molecular mass of the intact protein and to reveal any heterogeneity that would not be evident in SDS-PAGE analysis. The results are compared with calculated mass of the protein with the assumption that all the cysteines are disulphide-linked. The different peaks represent different charge states of the protein.
Result: Reverse phase chromatogram shows single sharp peak showing that the protein is pure and homogeneous.
Protein purity and structural homogeneity is analysed by reversed phase chromatography. 50 µg of protein, at 0.1 mg/ml in 10 mM HCl is analysed in ACE C4 4.6 x 250 mm column using eluted using a 10 – 90 % acetonitrile gradient in 0.1 % trifluoroacetic acid . Homogeneity is judged by the absence of multiple peaks and by the symmetry of the main peak. Blue line shows absorbance at 280 nm and the green line the acetonitrile gradient.
Result: UV spectrum shows full recovery of protein following aliquoting and lyophilisation.
Absorbance at 280 nm: average 0.180
Recovered concentration: 0.180 cm-1 x 10 / 0.76 cm-1 mg ml-1 = 1.18 mg / ml
Recovery: 110% (>100% due to routine 10% over-fill of vials during aliquoting)
The sample was reconstituted in 10 mM HCl to a theoretical concentration of 1 mg/ml following instructions above. This was diluted 1:10 in 6 M guanidine hydrochloride, 20 mM sodium phosphate pH 7.4 and the UV spectrum 340-220 nm. Concentration was calculated using extinction coefficient at 280 nm.
Result: Endotoxin level <0.005 EU/ug protein (below level of detection)
Stem cell cultures are sensitive to endotoxins1, which can be present in media, serum and as a contaminant on plasticware. We optimise our protein production processes to ensure the lowest possible levels of endotoxin contamination. Our endotoxin pass criteria are set at the industry leading <0.1 EU per ug protein and we aim for <0.01 EU per ug protein. Endotoxin levels in our proteins are determined by an external expert microbiological testing services provider.