Fel d 1 recombinant protein

Recombinant allergen Fel d 1 for Felis domesticus (cat)

Cat. Num. AAA505101

• Applications: ELISA
• Purity: Purity is >95% as determined by gel electrophoresis.
• Synonyms: Fel d 1; Recombinant allergen Fel d 1 for Felis domesticus (cat); Fel d 1 recombinant protein

• Host: Pichia pastoris
• Purity/Purification: Purity is >95% as determined by gel electrophoresis.
• Form/Format: Lyophilized from 20 mM HEPES buffer pH 8, 0.15 M NaCl and 5 mM EDTA

• Applicable Applications for Fel d 1 recombinant protein: ELISA (EIA)
• Application Notes: The evaluation of the recombinant allergen has been evaluated in an external study carried out at a Spanish hospital by a group of allergists with positive and negative serum samples from patients. The evaluation of the recombinant allergens has been performed by means of an in-house ELISA assay. In this immunoassay, it has been determined the presence of specific IgG in sera that had previously been validated by skin prick testing (SPT) and the UniCAP® test. The sera panel for this study was composed of 25 positive and 10 negative specimen sera.; ; The recombinant allergen Fel d 1 detected 22 positive sera out of 25 (82% incidence) with higher prevalence of sera with titers of 0.70-3.5 IU/ml (international unites per milliliter; 1 IU is equivalent to 2.42 ng of IgG). ; ; Possible Applications: Western Blot (WB), Dot Blot (DB), Indirect ELISA, Positive Control in Direct ELISA, Chemiluminescence Immunoassays (CLIA), Lateral Flow (LF). Where this product has not been tested for use in a particular technique, this does not necessarily include its use in such procedures. Suggested working dilutions are given as a guide only. It is recommended that the user titrates. The lab strongly recommends utilizing anti-CCD (cross-reactive-carbohydrate determinants) absorbent to eliminate the anti-CCD IgE antibodies present in normal human sera to prevent cross-reaction of this glycoprotein. By using this absorbent, reactions resulting from CCDs are stopped. In the specific case, you measure IgG with an allergen produced in P. pastoris, the lab recommends using an anti-CCD sorbent (Please Inquire) to avoid cross-reaction with antibodies in sera which react with fungal high mannose glycans.
• Presentation: Dry Powder
• Batch Composition: - Components: his-Fel d 1; - Composition: recombinant allergen with a his-tag
• Molecular Weight Note: Determined by SDS-PAGE, the protein shows a smear between molecular markers of 18,400 Da and 14,400 Da which corresponds with the monomer. The smear appearance might be due to the glycosylation pattern.
• Protein Concentration Determined Spectrophotometrically: DO₂₈₀ = 0.877; A_0.1% (=1 g/l) = 0.593; Concentration*: 1.48 mg/mL; ; *The measurement of the protein concentration has been performed with the theoretical extinction coefficient of the recombinant protein obtained from Gill and vonHippel, 1989. It is recommended that the users carry out their absorbance determinations to avoid equipment variabilities regarding final concentration, mainly in reproducibility analysis.
• Absence of Precipitation After a Freezing and Thawing Cycle: OK
• Reconstitution: With approx. 0.59 mL of sterile double-distilled water, a final concentration of approx. 1.48 mg/ml will be obtained. The solubilization of the cake should be developed for 15 min to allow a homogenous protein solution, considering that part of the cake can be on the glass-walls of the container. Upon reconstitution, leave the solution at least 15 min homogenizing with a mild agitation at 4°C. Avoid vigorous shaking that can cause foaming and protein denaturation. With this reconstitution, the protein will be maintained at pH 8. It is recommended that the users carry our their absorbance determinations to avoid concentration variabilities due to the equipment used, mainly in reproducibility analysis.
• Observations: Proteins should be maintained frozen at high concentrations. In order to defrost the protein, maintain the aliquot at 25°C without shaking to avoid aggregation. Prior to making test dilutions and after defrosting the protein, it is recommended to remove possible protein aggregates by centrifuging the stock solution, avoiding alterations in the immobilization of the biomolecule to the solid surface.
• Preparation and Storage: Upon receipt, store at 4°C or -20°C. Once reconstituted, it should be aliquoted in order to avoid repeated freezing and thawing cycles and stored at -20°C to -80°C.

: SDS-PAGE

(Purity Control in SDS-PAGE: 15% // Figure 1. SDS-PAGE analysis (15%) of 10 ul of recombinant allergen. Purity is >95% as determined by gel electrophoresis.)

Related Product Information for Fel d 1 recombinant protein

The Felis domesticus uteroglobin Fel d 1 has been prepared as the recombinant mature protein composed of two chains and fused to a his-tag.

References

Ohman, J. L., Lowell, F. C., Bloch, K. J. and Kendall, S. Allergens of mammalian origin V. properties of extracts derived from domestic cat. 1976, Clin. Allergy 6:419-28.
Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem. 1989 Nov 1;182(2):319-26.