Western Blot Antibody Customer Review for ADNP Polyclonal Antibody (STJ91502)


Target information

ADNP (Activity-Dependent Neuroprotector Homeobox) is expressed through the ADNP gene in humans. It is a possible transcription factor through having a homeobox and 9 zinc finger domains. It is said to have both stimulatory and inhibitory growth effects on different tumour cells. The ADNP Polyclonal Antibody detects endogenous levels of ADNP protein.

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1. HOW DID OUR ANTIBODY PERFORM? ANTIBODY CUSTOMER REVIEW: ADNP Polyclonal Antibody (STJ91502)
2. What was used? Primary antibody: STJ91502 ADNP Antibody Provider: St John’s Laboratory Ltd. Dilution ratio: 1:1000, 1:5000, 1:25000 Application: Western Blot Materials for validation: Hela cell lysate, HEK 293T cell lysate and EBV-infected lymphoblastoid cell lysate Click for product data sheet PDF
3. What Was The Protocol? Treatment of Materials Cells were cultured to 70%-80% plating density and lysed in RIPA buffer (Thermo Scientific) with protease inhibitors (Thermo Scientific) and phosphatase inhibitors (Roche). Equal volumes of 4X NuPage LDS sample buffer and 10X NuPage reducing agent (Life Technologies) were added for protein denaturing at 95¡ãC for 10 minutes. Gel Electrophoresis (SDS PAGE) Equal volumes (20ul) of protein samples with equal protein concentrations were loaded on a 4-12% Bis- Tris gel and run at constant voltage 200 V for 1.5h. Transfer 0.1 um nitrocellulose membrane (GE Healthcare, Life Sciences), semi-dry transfer in Pierce G2 Fast Blotter at constant current of 1.2 A for 10 minutes. Blocking 1 hour at room temperature in a solution of 5% non-fat dry milk in 1X TBST. Primary Antibody Incubation ADNP antibody was diluted to a concentration of 1:1000, 1:5000 and 1:25,000 in a solution of 5% non-fat dry milk in 1X TBST. The membrane was incubated with primary antibody overnight at 4¡ãC or for 1.5 hours at room temperature. Membrane Wash Membrane was washed with 1X TBST 4 times. Secondary Antibody Incubation HRP-labelled goat anti-rabbit (Biorad) was diluted to a final concentration of 1:10,000 in a solution of 5% non-fat dry milk in 1X TBST. The membrane was incubated with the secondary antibody for 1 hour at room temperature. Membrane Wash Membrane was washed with 1X TBST 4 times. Visualization HRP-labeled goat anti-rabbit (Biorad) was diluted to a final concentration of 1:10.000 in a solution of 5% non-fat dry milk in 1X TBST. The membrane was incubated with the secondary antibody for 1h at room temperature.
4. Experiment 1 Gel electrophoresis information: 4-12% Bis-Tris gel, constant voltage 200V, 1.5 hours Transfer information: 0.1 um nitrocellulose membrane, semi-dry fast blotting, 10 minutes at constant current of 1.2 A Visualization information: ECL Plus western blotting substrate No. Antigen Loading amount Primary antibody Primary antibody dilution ratio Secondary antibody Target band KD Visualization time 1 Hela cell lysate 6.92 ug STJ91502 ADNP antibody 1:1000 HRP labelled goat anti-rabbit (Biorad) ~124 KD 1 minute exposure intervals 10 minutes total visualization time 2 HEK 293T cell lysate 15 ug 3 EBV-infected lymphoblastoid cells 15 ug 4 Hela cell lysate 6.92 ug 1:50005 HEK 293T cell lysate 15 ug 6 EBV-infected lymphoblastoid cells 15 ug 7 Hela cell lysate 6.92 ug 1:25,0008 HEK 293T cell lysate 15 ug 9 EBV-infected lymphoblastoid cells 15 ug
5. What were the results? Experiment 1: Bands are detected between 65- 115 kDa. Since ADNP is expected to be a ~124 kDa protein based on mRNA data, these bands all seem to be non-specific for ADNP. A remark that should be made is that the lanes with HEK (lanes 2,5 and 8) and EBV-infected lymphoblastoid (lanes 3, 6 and 9) cell lysates don¡¯t seem to migrate properly due to a too high protein concentration (15 ug) loaded. This will be taken into account in experiment 2.
6. Experiment 2 Gel electrophoresis information: 4-12% Bis-Tris gel, constant voltage 200V, 1.5 hours Transfer information: 0.1 um nitrocellulose membrane, semi-dry fast blotting, 10 minutes at constant current of 1.2 A Visualization information: ECL Plus western blotting substrate No. Antigen Loading amount Primary antibody Primary antibody dilution ratio Secondary antibody Target band KD Visualization time 1 Hela cell lysate 6 ug STJ91502 ADNP antibody 1:1000 HRP labelled goat anti-rabbit (Biorad) ~124 KD 1 minute exposure intervals 5 minutes total visualization time 2 HEK 293T cell lysate 3 EBV-infected lymphoblastoid cells 4 Hela cell lysate Actin-beta antibody (Abcam) 1:5000 HRP labelled rabbit anti-mouse ~42 KD5 HEK 293T cell lysate 6 EBV-infected lymphoblastoid cells
7. What were the results? Experiment 2: Antigen detection against actin-beta was used as an internal control to assess the western blotting method. A signal at ~ 42 kDa at the expected molecular weight of actin-beta can clearly be observed. At first, this made us suggest that the antibody tested for ADNP detection is non-specific or at least not able to detect ADNP in the materials of our research interest. Despite this, exploring literature showed us that different antibodies for ADNP detection rendered multiple bands in a range of ¡À 115-180 kDa (Furman et al. 2004; Gennet et al. 2008). Since we observed a signal around ~ 185 kDa, we decided that our acquiring technique might have missed out on the specific ADNP signal due to sensitivity. For experiment 3 we therefore switched from the ECL Plus western blotting substrate to the ECL Femto substrate, which is said to be more sensitive.
8. Experiment 3 Gel electrophoresis information: 4-12% Bis-Tris gel, constant voltage 200V, 1.5 hours Transfer information: 0.1 um nitrocellulose membrane, semi-dry fast blotting, 10 minutes at constant current of 1.2 A Visualization information: ECL Femto western blotting substrate No. Antigen Loading amount Primary antibody Primary antibody dilution ratio Secondary antibody Target band KD Visualization time 1 Hela cell lysate 6 ug STJ91502 ADNP antibody 1:1000 HRP labelled goat anti-rabbit (Biorad) ~124 KD but multiple bands possible around ¡À 115-180 kDa 1 minute exposure intervals 15 minutes total visualization time (but bands already clearly visible after 2 min visualization) 2 HEK 293T cell lysate 3 EBV-infected lymphoblastoid cells
9. What were the results? Experiment 3: After switching to the more sensitive ECL Femto western blotting substrate, we could observe multiple faint bands between ¡À 115-180 kDa.
10. What did the customer think? Antibody Specificity: Antibody Rating: Testing results were provided independently by E. Cappyuns at the University of Antwerp. They stated ¡°Based on a parallel experiment with an LSBio ADNP antibody and the corresponding blocking peptide, we could determine that using the St. John¡¯s Laboratory ADNP antibody renders a specific signal for ADNP amongst non-specific bands. Since similar observations were mentioned in the literature, we report this antibody to be a good antibody for the detection of ADNP, however not as specific as expected from the supplied data sheet.¡±
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