Recombinant Rat SI Protein, His (Fc)-Avi-tagged

• Cat.No.: SI-5055R
• Product Overview: Recombinant Rat SI with His (Fc)-Avi tag was expressed and purified

Specification

• Species: Rat
• Source: HEK293
• Tag: Avi&Fc&His
• Endotoxin: < 1.0 EU per μg of the protein as determined by the LAL method
• Purity: ≥85% by SDS-PAGE
• Stability: Stable for at least 6 months from the date of receipt of the product under proper storage and handling conditions. Avoid repeated freeze-thaw cycles.
• Storage: For long term storage, aliquot and store at -20 to -80 centigrade. Avoid repeated freezing and thawing cycles.
• Storage Buffer: PBS buffer

Gene Information

• Gene Name: Si sucrase-isomaltase (alpha-glucosidase) [ Rattus norvegicus ]
• Official Symbol: SI
• Gene ID: 497756
• mRNA Refseq: NM_013061.1
• Protein Refseq: NP_037193.1
• UniProt ID: P23739

Q&As

There is a weak signal in lane 1 at a somewhat higher MW of ~130 kD for SI-0515H. I presume this is the actual enzyme which appears as a ~130 kD protein. This was also observed in the COA for SI-1516H, as shown below, but with a much higher signal. The concentrations of SI-0515H and SI-0516H are 0.12 mg/mL and 0.38 mg/mL, respectively. The signal in the Western blots do not scale comparably for the two enzymes. Am I reading these WB correctly, and if so, what may could account for what appears to be >50-fold differences in signal whereas there only a ~3-fold difference in protein concentration?

The strength of the WB band signal is not only related to the protein concentration, but also related to the binding affinity between protein and the antibody. Due to the differences in structure, SI-0515H and SI-0516H could have different binding affinity to the antibody, thus the WB results are different.

Would it be fair to say that the higher MW band of ~130kD in Lane 1 for both enzymes is likely due to glycosylation?

Yes, it is likely that the band shifting was due to glycosylation.

I see that the expression system was changed from baculovirus to HEK293 from the samples provided back in April compared with the two recent samples. The WB from the samples in April are shown below, which shows a primary signal at ~105 kD, along with some minor bands at 70-80 kD. Was the shift the HEK293 due to the fact that only a single band showed up in the WB?

Different expression system has different post-translational modification. The SI proteins expressed from HEK293 were in soluble form, which is more close to its native status.