Validation Report #029802

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BCL2-Associated Agonist of Cell Death (BAD) antibody (ABIN674709) 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: #029802
Validated on: 08/26/14

Summary

Antigen

BCL2-Associated Agonist of Cell Death (BAD)

Catalog number
Supplier

Bioss

Supplier catalog number
Lot number

090915

Method validated
Laboratory
Validation number
Positive Control
Negative Control
Notes

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

Independent Results

Full Methods

Primary Antibody

  • Antigen: BCL2-Associated Agonist of Cell Death (BAD)
  • Catalog number: ABIN674709
  • Supplier: Bioss
  • Supplier catalog number: bs-1304R
  • Lot number: 090915
  • Antibody Dilution: 1:200

Loading Control Antibody

  • Antigen: Mouse Anti-Actin
  • Supplier: BD Transduction Laboratories
  • Catalog number: 612657
  • Antibody Dilution: 1:6,000

Secondary Antibody

  • Antigen: Goat Anti-Rabbit IgG (H + L)-HRP Conjugate
  • Supplier: Bio-Rad
  • Catalog number: #170-6515
  • Lot number: L170-6515
  • Antibody Dilution: 1:10,000

Controls

  • Positive control: MCF7 cells
  • Negative control: C6/36 cells

Protocol

  1. The cell extracts were heated at 95°C for 5 minutes in 1X SDS Sample Buffer containing 1% SDS and 1.25% β-mercaptoethanol.
  2. 15 μl of heated 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 hour.
  6. The membrane was rinsed with TBST once.
  7. The membrane was immersed with the protein side up in the primary antibody solution in TBST containing 5% (W/V) BSA and incubated for 16 hours at 4°C.
  8. The membrane was rinsed in TBST thrice for 5 minutes each.
  9. The membrane was incubated in the HRP-conjugated secondary antibody solution 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 minutes each.
  11. The membrane was rinsed in TBS twice for 30 seconds each.
  12. Signals were detected with ECL-2 Substrate. The blot was scanned for 600 seconds.
  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 minutes each.
  15. The membrane was washed in TBST 2 times for 10 minutes each.
  16. Repeated Steps 5-12 with the loading control antibody (for Anti-actin) and its matching secondary antibody.

Experimental Notes

  • No experimental challenges noted.

Figures

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Figure 1: Western Blot for BAD. Arrowhead indicates the expected molecular weight of ~18 kDa.
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