Detection of a functionally deficient AAT protein variant by protease inhibitor (PI) typing
Alpha-1-antitrypsin deficiency (AATD) is a rare hereditary condition resulting from the mutations in the SERPINA1 (serine protease inhibitor) gene and is characterized by low circulating levels of the alpha-1 antitrypsin (AAT) protein. The traditional algorithm for laboratory testing of AATD involves the analysis of AAT concentrations (nephelometry), phenotyping (isoelectric focusing, IEF), and genotyping (polymerase chain reaction, PCR).
For IEF the ampholyte HR 4.2-4.9 is required. Did you know that Isogen Life Science is the only provider for this ampholyte?
If you are struggling with your assays and need customised ampholytes, we can help you with this request.
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Both HR and SH CEInfinite AESlytes are good for iCIEF of monoclonal antibodies (mAbs) (see below figure), fusion proteins, antibody drug conjugates (ADCs), dairy proteins, meat proteins and seed proteins.
SH CEInfinite AESlytes are especially good for iCIEF of acidic fusion proteins (see below figure), dairy proteins, meat proteins and seed proteins; while HR CEInfinite AESlytes are notably good for neutral and basic fusion proteins.
The separation efficiency of some ADCs is improved when mixing SH and HR CEInfinite AESlytes (see below figure).
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UH CEInfinite AESlytes are specially designed for separation of fusion proteins and other complex proteins.
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CEInfinite AESlytes (HR, SH and UH) are provided at broad-range and narrow range pH intervals. Broad range CEInfinite AESlytes allow for a universal iCIEF method for a range of proteins with different isoelectric points and delivers fast focusing of proteins. Narrow range CEInfinite AESlytes provide higher separation efficiency at the cost of slower focusing of proteins (see below figure). Specific iCIEF methods have to be developed when the pI of proteins is in different ranges.
CEInfinite AESlytes have demonstrated superior focusing and low UV noise. The below figure illustrated focusing of 6 pI markers with CEInfinite AESlyte SH pH 4-8 and pH 3-7 from another vendor. Much higher baseline noise was observed from the electropherogram with CAs from the other vendor. Furthermore the pI 5.85 did not complete focusing with CAs from the other vendor under the same experimental conditions.
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CEInfinite AESlytes allow direct coupling of iCIEF to a mass spectrometer (MS). CEInfinite AESlytes are developed to be MS compatible (contain low salt) and have an extremely high percentage of good amphoteric components.
High protein iCIEF resolution can be achieved even with very low AESlyte concentration (0.5%). These advantages greatly minimise possible interference of MS signal by salts and CAs.
In addition, “widened” protein peaks with narrow range CAs help with mobilisation of focused protein peaks into the transfer capillary, further reducing protein remixing.
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The CEInfinite Preparative instrument allows you also to perform offline iCIEF-MS by protein fractionation. The collected fractions can be further analysed with MS.
We understand the complexity of a protein. To achieve higher resolution and better separation of your current (i)CIEF assays, we also offer custom made AESlytes CAs upon request. Please contact us for further support on this custom-made solution services.
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