Product Review: TAZ Antibody (AAS99633C) in mouse cardiac mitochondria lysate using Western blot

Product Page for TAZ Antibody-C-terminal region (AAS99633C)

Researcher:Corey Powers, Cincinati Children’s Hospital Medical Center
Application:Western blotting
Species+tissue/cell type:
Lane 1: 20ug mouse cardiac mitochondria lysate
Lane 2: 20ug mouse cardiac mitochondria lysate
Lane 3: 20ug mouse cardiac mitochondria lysate
Lane 4: 20ug mouse cardiac mitochondria lysate
Primary antibody dilution:1:500
Secondary antibody:Goat anti-rabbit-HRP
Secondary antibody dilution:1:2500

Questionnaire:
How do Antibody Verify’s reagents play a role in your experimental goals? Tafazzin is a target gene in our research, thus a solid antibody would enbale further analysis for us.
Would you use this antibody in future experiments? If was improved, yes.
If the antibody works, do you plan to use it in future experiments or to publish your data? Why or why not? We would like to use it, however more antibody would be required to validate the spotty results that I have gotten.
How did you store the antibody after re-suspension?  -20C in aliquots
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) Mus musculus 2) Cardiac mitochondria 3) 20ug protein/Lane
How many different experimental trials were conducted using the antibody sample? 4 trials
How was this sample prepared? Sample was lysed with KH2PO4 + 2% Triton X-100. Then added H20, loading buffer and a reducing agent. Then heated to 95C for 10 minutes prior to loading.
Primary antibody dilution and incubation time: 1:500 in 5% BSA + PBS-T, Overnight at 4C with a agitation.
Secondary antibody used and dilution and incubation time: Goat Anti-Rabbit-HRP 1:2500, 1 Hour at RT with 5% milk + PBS-T
What controls were used in your experiment (positive/negative)? We used a + Ab control, mMDH which shows up in all of our samples.
Please include your detailed WB Procedure/Protocol here: Lysed with KH2PO4 + 2% Triton X-100, incubate sample on ice 30 min.
Add 4X loading buffer, 10X Reducing agent, H2O and sample to 20ul total volume. Protein was added to ensure 20ug in each lane (40ug total).
Sample were heated to 95C for 10 minutes and then iced until loading. We use the invitrogen Novex system. Thus we use a 1x MOPS running buffer and 1x MOPS in the inner chamber of the gel chamber with 500ul of Antioxidant. We used a 4-12% Bis-Tris Gel. The gel was run at 120V for ~2 hours. We then used PVDF membrane and a semi-dry transfer unit to transfer for 30 minutes at 25V. We washed the membrane in 0.5% PBS-Tween20 3×15 minutes and then blocked overnight at 4C in 5% BSA+PBS-T. We then washed 3×15 minutes in PBS-T. Wash 3×15 minutes w/ PBS-T, incubate for 1 Hour with secondary Goat-Anti-Rabbit HRP 1:2500 in 5% milk + PBS-T. Wash 3×15 minutes with PBS-T and then detect using a GE STORM with GE ECL Prime with a 1:1 ratio of the reagents for a total of 1ml.
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