Validation Report #029849

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Glutathione S Transferase (GST) antibody (ABIN1998462 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: #029849
Validated on: 11/06/15

Summary

Antigen

Glutathione S Transferase (GST)

Catalog number
Supplier

Sino Biological

Supplier catalog number
Lot number

HD04FE1006

Method validated
Laboratory
Validation number
Positive Control

GST- AcNFkBp(C-terminal) 293-Lysate

Negative Control

Empty vector- 293 Lysate

Notes

A strong specific band was observed in the positive control at the expected size (~82 kDa) that is not observed in the negative control.

Independent Results

Full Methods

Primary Antibody

  • Antigen: Glutathione S Transferase (GST) protein
  • Catalog number: ABIN1998462
  • Supplier: Sino Biological
  • Supplier catalog number: 11213-MM01
  • Lot number: HD04FE1006
  • Antibody Dilution: 1:7000

Loading Control Antibody

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

Secondary Antibody

  • Antigen: Goat Anti-Mouse IgG (H + L)-HRP Conjugate
  • Supplier: Bio-Rad
  • Catalog number: #170-5047
  • Lot number: N/A
  • Antibody Dilution: 1:15,000

Controls

  • Positive control: GST- AcNFkBp(C-terminal) 293-Lysate
  • Negative control: Empty vector- 293 Lysate

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 were loaded and resolved on 8-16% SDS-polyacrylamide gel.
  3. The Bio-Rad Precision Plus (Cat 161-0374) were used as molecular mass markers.
  4. Proteins were then transferred onto PVDF membrane by wet transfer.
  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 24 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 1 minute.
  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

None reported

Figures

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Figure 1. Probing of cell extracts with GST Tag (upper panel) or loading control Actin (lower panel) antibodies. Lane 1: GST-AcNFkBp(C-terminal)293-Lysate; Lane 2: Empty vector - 293 Lysate
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