vWF (Recombinant Fragment)

Frequently Asked Questions

What is the expected molecular weight of vWF (Recombinant Fragment) on SDS-PAGE or Western blot?

Because this is a recombinant fragment of the full-length 2,813 aa vWF precursor (UniProt P04275) expressed in HEK293 cells, the apparent molecular weight on SDS-PAGE will be larger than the predicted polypeptide backbone due to extensive N- and O-linked glycosylation. Expect a diffuse band or smear consistent with heterogeneous glycoforms — this is normal and expected for HEK293-expressed vWF material. Running under reducing conditions and comparing against a broad-range MW ladder (10–250 kDa) is recommended. A sharp, discrete band would suggest incomplete glycosylation and should be treated as a quality concern.

Which domain or processing form of vWF does this recombinant fragment correspond to?

This product is a defined recombinant fragment of human vWF, not the full-length mature protein or the pro-vWF precursor. The full-length vWF undergoes signal peptide cleavage, propeptide removal, and multimerization before secretion. This fragment is expressed in HEK293 cells, preserving the authentic glycosylation profile of the relevant domain(s). It is intended as a structurally native antigen for ELISA standardization and antibody validation, rather than as a model of the full multimeric plasma form. Confirm the specific fragment boundaries in the certificate of analysis included with each lot.

What cleaves vWF in vivo and is this recombinant fragment a substrate for ADAMTS13 activity assays?

In vivo, vWF is cleaved by ADAMTS13 at the Tyr1605–Met1606 bond within the A2 domain, a cleavage event central to regulating ultra-large vWF multimer size and preventing thrombotic microangiopathy. Whether this recombinant fragment serves as a productive ADAMTS13 substrate depends on whether the A2 domain cleavage site is present in the expressed fragment. Check the certificate of analysis for domain coverage. If the A2 site is included, activity can be assessed by SDS-PAGE or FRETS-vWF73 analog assays under denaturing or fluid-shear conditions at pH 7.8, 37°C.

What buffer conditions are recommended for vWF fragment binding assays such as GPIbα or collagen binding ELISAs?

The product is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. For plate-based binding assays (e.g., GPIbα or collagen binding ELISA), dilute into assay buffer such as PBS pH 7.4 with 0.1% BSA prior to coating or incubation — the glycerol concentration should be reduced to ≤1% in the final assay volume to avoid interference with protein adsorption. For ristocetin-induced GPIb binding assays, supplement with 1.5 mg/mL ristocetin and maintain Ca²⁺ at physiological concentrations. Avoid repeated dilution cycles from the same aliquot; single-use aliquots are supplied to prevent activity loss.

What starting concentration of recombinant vWF fragment should I use when building an ELISA standard curve?

For sandwich ELISA standardization, a top standard of 10–100 ng/mL is a reasonable starting point, with a 2-fold or 3-fold serial dilution across 7–8 points down to 0.1–0.5 ng/mL, depending on your detection antibody sensitivity. Protein concentration is determined by A280 and validated by SDS-PAGE densitometry (>95% purity). Because this fragment carries authentic HEK293 glycosylation, it more accurately reflects the epitope presentation of endogenous plasma vWF than bacterially expressed alternatives, improving standard curve linearity when paired with antibodies raised against native vWF.

Can I use REC-vWF as a positive control for Western blot with the matched RP-vWF rabbit polyclonal antibody?

Yes — REC-vWF is the recommended positive control for Western blot using RP-vWF (/anti-vwf-rabbit-polyclonal-antibody). Because both products are developed in the same lab against the same antigen, epitope compatibility is guaranteed. Load 50–200 ng of REC-vWF per lane under reducing SDS-PAGE conditions alongside your cell lysate or plasma sample. Expect detection of the glycosylated fragment band with RP-vWF at a primary antibody dilution of 1:500–1:2,000 (optimize per your secondary and detection system). This pairing is particularly useful for confirming antibody lot-to-lot consistency during assay validation.

How much recombinant vWF (Recombinant Fragment) should I load for Western blot positive control?

Load 50–200 ng per lane as a starting range. At 100 ng under reducing conditions, RP-vWF (SKU: RP-vWF) reliably detects the vWF fragment band without signal saturation on standard chemiluminescent systems. If you are titrating your antibody or validating a new lot, run a 3-point dilution series — 50, 100, and 200 ng — in parallel with your experimental lanes. Given the diffuse banding expected from glycosylated HEK293-expressed material, ensure adequate transfer time when using PVDF membranes, particularly for higher-MW glycoforms.

How should I store and handle the vWF recombinant fragment to maintain activity across multiple experiments?

Store at -20°C in single-use aliquots immediately upon receipt. The supplied buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — is formulated to maintain stability during freeze-thaw, but repeated freeze-thaw cycles progressively reduce binding activity and increase aggregation risk, which is especially problematic for a glycoprotein prone to non-native multimerization. Do not store working dilutions at 4°C for more than 24–48 hours. If your experiment requires multiple days of use, prepare fresh dilutions from separate aliquots rather than re-freezing partially used stock.