Validation Report #029759

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Caspase 8, Apoptosis-Related Cysteine Peptidase (CASP8) antibody (ABIN724205) 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: #029759
Validated on: 07/02/14

Summary

Antigen

Caspase 8, Apoptosis-Related Cysteine Peptidase (CASP8)

Catalog number
Supplier

Bioss

Supplier catalog number
Lot number

131127

Method validated
Laboratory
Validation number
Positive Control
Negative Control

c6/36 mosquito cells (non-reactive species)

Notes

A strong band was observed at the correct molecular weight in the positive control sample. No major bands were observed in the negative sample.

Independent Results

Full Methods

Primary Antibody

  • Antigen: Caspase 8, Apoptosis-Related Cysteine Peptidase (CASP8) (1:200 dilution)
  • Catalog number: ABIN724205
  • Supplier: Bioss
  • Supplier catalog number: bs-0052R
  • Lot number: 131127

Loading Control Antibody

  • Antigen: Mouse Anti-Actin (1:6,000 dilution)
  • Supplier: BD Transduction Laboratories
  • Catalog number: 612657
  • Lot number: N/A

Secondary Antibody

  • Antigen: 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: HeLa cell extract
  • Negative control: c6/36 cell extract

Protocol

  1. 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.
  2. 20 μg of boiled extracts were loaded and resolved on 8-16% SDS-polyacrylamide gel.
  3. The Thermo Scientific - Spectra Multicolor Broad Range (Cat # 26634) were used as molecular mass markers.
  4. Proteins were then transferred onto PVDF membrane by wet transfer and protein transfer was confirmed with Ponceau-S staining.
  5. 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) BSA at room temperature for 1 h.
  6. The membrane was rinsed with TBST once.
  7. The membrane was immersed with the protein side up in the primary antibody solution (CASP8; 1:200) in TBST containing 5% (W/V) BSA and incubated for 16 h at 4°C.
  8. The membrane was rinsed in TBST thrice for 5 min each.
  9. 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) BSA and incubated for 1 hour at room temperature (~26°C) with gentle agitation.
  10. The membrane was rinsed thrice TBST thrice for 5 min each.
  11. The membrane was rinsed in TBS twice for 30 s each.
  12. Signals were detected with ECL-2 Substrate. The blot was scanned for 300 s.
  13. The membrane was rinsed three times TBST.
  14. Incubated in Acidic Glycine Stripping Buffer at room temperature with gentle agitation for 3 times, 10 min each.
  15. The membrane was washed in TBST 2 times for 10 min each.
  16. 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

  • Nothing to note.

Figures

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Figure 1: Western blot of lysates from HeLa cells (Lane 1), and c6/36 cells (Lane 2) probed with anti-CASP8 (upper panel) or with anti-Actin for loading control (lower panel).
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