Product Review: NR4A2 antibody – N-terminal region (ARP38753_P050) in Nuclear fraction from mouse cortex using Western Blot

Product Page for NR4A2 antibody – N-terminal region (ARP38753_P050)

Researcher: Sorce Silvia, University of Zurich
Application: Western Blotting
Species+tissue/cell type: Lane 1: Nuclear fraction from mouse cortex
Primary antibody dilution: 1:400
Secondary antibody: Goat anti rabbit-HRP
Secondary antibody dilution: 1:10,000

 

ARP38753-QC10819-WB-image-02

 

 

 

 

 

 

 

 

Questionnaire:
How do Aviva’s reagents play a role in your experimental goals? It is really important for us to distinguish the human and murine Nr4a2 proteins
Would you use this antibody in future experiment? For our project we would need a more specific antibody to distinguish the human and murine proteins
Have you used another antibody which has worked in your application? No
If the antibody works, do you plan to use it in future experiments or to publish your data? Why or why not? Probably, but for the moment we are going to use in situ hybridization.
How did you store the antibody after re-suspension? We aliquoted the antibody and stored it at -20°C.
Sample Description (please include 1) species type, and 2) cell/tissue type, and 3) how much protein loaded for each lane of your gel): 1) Mouse 2) nuclear subcellular fractions 3) Nuclear fraction: 5x volume/weight, 15 ul of sample per lane
How many different experimental trials were conducted using the antibody sample? One trial.
How was this sample prepared? Fractionation: Tissue was homogenized gently in a hypotonic buffer based on Hepes by applying 10 strikes with a micro pestle and swelling for 10 min. Isotonic conditions were restored by the addition of 25 mM sucrose. Nuclei were centrifuged for 10 min at 800 g and dissolved in common RIPA buffer. Sample preparation was performed using a 4x NuPAGE® LDS Sample Buffer (Invitrogen) by cooking at 70°C for 10 min and subsequent centrifugation. Supernatant was submitted to electrophoresis.
Primary antibody dilution and incubation time: 1:400 in 1x TBS-T overnight at 4°C
Secondary antibody used and dilution and incubation time: 1:10000 goat anti-rabbit IgG in 1xTBS-T 1 h at room temperature
What controls were used in your experiment (positive/negative)? No negative controls were used.
Please include your detailed WB Procedure/Protocol here: • SDS-Page was carried out using the 4x NuPAGE® LDS Sample Buffer for sample preparation, the 20x NuPAGE® MES SDS Running Buffer,
12% NuPAGE® Bis-Tris Precast gels and the XCell SureLockTM gel electrophoresis system (All Invitrogen) according to the manufacturer’s
manual.
• Electrophoretic separation was conducted for 2 – 2.5 h at 100V.
• Prepare 1x Transfer buffer solution with 20% methanol and mix thoroughly (10 mM Tris pH 7.6, 100 mM NaCl, 0.01% Tween-20)
• Prepare Western Blot Full tank system from Bio-Rad and assemble as mentioned in the manufacturer’s manual. Nitrocellulose membrane is
pre-incubated in ready-Transfer buffer containing methanol.
• Remove all air bubbles and close the blotting cassette firmly, being careful not to move the gel and the filter paper sandwich
• Place the cassettes in the module, add the ice-cooling unit and fill the chamber completely with Transfer buffer
• Run the blot for 1.5 h at 75 V, take care to reduce dissipation heat
• Upon completion of the run, rinse blot in water and block membrane n 5% Blocking solution in 1x PBS-T for 1 h at room temp on a lab shaker
• Replace with fresh 10 ml of 1% Blocking solution in 1x TBS-T containing the primary antibody in the appropriate dilution and incubate O/V at 4°C
on a shaker
• Wash 3 times with 1x TBS-T with 1% Blocking for 10 min each time at room temp
• Add 10 ml of 1% Blocking solution in 1x TBS-T containing the HRP-conjugated secondary antibody (Dilute: 1:10’000) and incubate 1 h at room
temp
• Wash 4 times with 1x TBS-T for 15 min each time
• Develop membrane 5 min in ECL subtrate (Millipore) and record picture using a CCD camera of the Stella Imaging System (raytest
Isotopenmessgeräte GmbH, Straubenhardt, Germany)
How would you rate this antibody on a scale from 1-5 (5=best) and why? 3, We detected a band at the proposed site but we also developed unspecific bands.
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