Validation Report

#029750

Insulin-Like Growth Factor 1 Receptor (IGF1R) antibody (ABIN726575) has been independently validated for Western Blot by the Science Exchange network of experts.

The Science Exchange network is used to independently validate research reagents, protocols, and results to ensure accuracy and reproducibility.

Validation number: #029750

Validated on: 07/01/14

Summary

Antigen

Insulin-Like Growth Factor 1 Receptor (IGF1R)

Catalog number

Supplier

Bioss

Supplier catalog number

Lot number

130322

Method validated

Laboratory

Validation number

Positive Control

MCF-7 cells – high expression

Negative Control

PC3 cells – low expression

Notes

Two strong bands were observed in the positive control, one at the expected molecular weight, and one slightly higher (which may represent a glycosylated or precursor form). Weaker bands of the same molecular weight appear in the negative control, which is consistent with the expected lower expression in PC3 cells. No other major bands are present in either positive or negative controls.

Independent Results

Full Methods

Primary Antibody
  • Antigen: Insulin-Like Growth Factor 1 Receptor (IGF1R) (1:200 dilution)
  • Catalog number: ABIN726575
  • Supplier: Bioss
  • Supplier catalog number: bs-0227R
  • Lot number: 130322
Loading Control Antibody
  • Antibody: Mouse Anti-Actin (1:6,000 dilution)
  • Supplier: BD Transduction Laboratories
  • Catalog number: 612657
  • Lot number: N/A
Secondary Antibody
  • Antibody: Goat Anti-Rabbit IgG (H + L)-HRP Conjugate (1:20,000 dilution)
  • Supplier: Bio-Rad
  • Catalog number: #170-6515
  • Lot number: L170-6515
Controls
  • Positive control: MCF-7 cell extract
  • Negative control: PC-3 cell extract
Protocol
  • Total protein extracts were boiled in 1X SDS Sample Buffer containing 1% SDS and 1.25% β-mercaptoethanol at 95°C for 5 min prior to loading.
  • 46 μg of boiled extracts were loaded and resolved on a 8-16% SDS-polyacrylamide gel.
  • The Spectra Multicolor Broad Range (Thermo Scientific, Cat # 26634) were used as molecular mass markers.
  • Proteins were transferred onto PVDF membrane by wet transfer and protein transfer was confirmed with Ponceau-S staining.
  • The PVDF membrane was incubated with 25 mL of blocking buffer [Tris Buffered Saline, pH 7.4 plus 0.1% TW20 (TBST)] containing 5% (W/V) non-fat dry milk at room temperature for 1 h.
  • The membrane was rinsed with TBST once.
  • The membrane was immersed with the protein side up in the primary antibody solution (anti-IGF1R; 1:200) in TBST containing 5% (W/V) non-fat dry milk and incubated for 16 hours at 4°C.
  • The membrane was rinsed in TBST three times for 5 min each.
  • The membrane was incubated in the HRP-conjugated secondary antibody solution (Goat anti-rabbit IgG-HRP; 1:20,000) in TBST containing 5% (W/V) non-fat dry milk and incubated for 1 hour at room temperature (~26°C) with gentle agitation.
  • The membrane was rinsed three times with TBST for 5 min each.
  • The membrane was rinsed in TBS twice for 30 s each.
  • Signals were detected with ECL-2 Substrate. The blot was scanned for 300 s.
  • The membrane was rinsed three times with TBST, then incubated in Acidic Glycine Stripping Buffer at room temperature with gentle agitation for 3 times, 10 min each.
  • The membrane was washed in TBST 2 times for 10 min each.
  • Repeated Steps 5-12 with the loading control antibody (anti-Actin; 1:6,000) and its matching secondary antibody (Goat anti-rabbit IgG-HRP; 1:20,000).
Experimental Notes
  • No challenges noted.

Figures

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Figure 1. Western blot of lysates from MCF7 cells (Lane 1) and PC-3 cells (Lane 2) probed with anti-IGF1R (upper panel) or with anti-Actin for loading control (lower panel).