Researcher: The University of Queensland, School of Medicine, Centre for Kidney Disease Research
Application: Western blotting
Species + Tissue/Cell type: Human HK2 cell (kidney proximal tubular cell line)
How many ug's of tissue/cell lysate run on the gel:
1. 40 ug HK2 cell (kidney proximal tubular cell line)
2. 40 ug H2O2 treated human HK2 Cell lysate
3. 40 ug H2O2 treated human HK2 Cell lysate
4. 40 ug H2O2 treated human HK2 Cell lysate
5. 40 ug H2O2 treated human HK2 Cell lysate
6. 40 ug H2O2 treated human HK2 Cell lysate
Primary antibody dilution: 1:1000
Secondary antibody: Goat anti-Rabbit HRP
Secondary antibody dilution: 1:2000
|How do Aviva's reagents play a role in your experimental goals?||Determine their expression levels in response to oxidative stress.|
|How would you rate this antibody on a scale from 1-5 (5=best) and why?||4.5 - very specific band found at 24 kDa.|
|Would you use this antibody in future experiments?||Yes|
|Have you used another antibody which has worked in your application?||Yes, but your antibody produced a cleaner result.|
|Do you believe the information about the reagent on Aviva's website is correct?||Yes|
|If the antibody works, do you plan to use it in future experiments or to publish your data? Why or why not?||Yes, SOD2 expresson is very important in oxidative stress research.|
|How did you store the antibody after re-suspension?||Aliquoted for single use at -20 degree Celsius.|
|Sample Description, Species, Tissue/Cell Type:||Human. HK2 cell (kidney proximal tubular cell line). 40 ug|
|How many different experimental trials were conducted using the antibody sample?||1|
|What type of experimental sample are you using and how did you preparing it?||HK2 cells were grown in 10cm cell culture dishes in Dulbecco's modified Eagle's Medium: Ham's F-12 (DMEM/F12) containing antibiotics Penicillin (1000 U/ml) and Streptomycin (1000 ug/ml), and fetal bovine serum (FBS) and allowed 24h to adehere. Cells were exposed to hydrogen peroxide (H2O2) diluted in growth media at 0.2, 0.4, 0.6. 0.8, 1.0 mM for 2h.|
|Primary used and dilution:||1:1000; overnight (approx 18h) at 4 degree celsius.|
|Secondary used and dilution:||Goat anti-rabbit IgG Horseradish peroxidate conjugate(G21234) (Molecular Probes-Invitrogen), 1:2000, 1h at RT.|
|What controls were used in your experiment? Please include your positive control:||Untreated cells.|
|Experimental Procedure/Protocols:||Cells were placed on ice and washed X3 with cold phosphate buffered saline (PBS), then scraped into radio-immunoprecipitation assay (RIPA) cell lysis buffer (0.15 M sodium chloride, 0.025 M sodium fluoride, 0.5 M ethylenediaminetetraacetic acid), 0.1% sodium dodecylsulphate (SDS), 1.0% lgepal in 50 mM Tris-Cl, pH 7.5) containing phosphatase and protease inhibitors (10 ug/mL aprotinin, 10 ug/mL leupeptin, 1mM sodium orthovanadate and 100 ug/mL phenylmethylsulfonyl chloride). After cell lysis by sonification, cell debris was removed by centrifugation (13,000 g, 20 min, 4 degree C). Protein concentration was determined using a Pierce bicinchoninic acid (BCA) protein assay and spectroscopy at 540 nm. Cell lysate was snap frozen in liquid nitrogen and stored at -80 degree c.40 ug of each protein were loaded into seperate lanes of a 10% sodium dodecylsulphate (SDS)-polyacrylamide gel and electrophoresed using Bio-Rad equipment, then electrophoretically transfered onto PVDF membrane. Bio-Rad Precision Plus Protein Kaleidoscope (Cat#161-0375) was routinely used as a protein size marker. To inhibit non-specific binding, membranes were first soaked in a blocking solution of 5 % skim milk in Tris-buffered saline (TBS) with Tween-20 pH 7.4 (TBST) (5% blotto) for 1 hour. Membrane were blotted with the primary antibody provided. Primary antibodies were diluted in a 1% blotto solution and incubated for 18-24 h at 4 degree C. Membranes were washed X3 in TBST for 5 min. Goat anti-rabbit IgG-HRP-conjugated secondary antibody diluted at 1:2000 in 1% blotto was used for 1h at RT. Protein bands were visualised using enhanced chemiluminescence and X-ray flim. Developed flims were scanned using a Canon Canoscan 8400F at 300dpi.|
Researcher: Manisha Nautiyal, Hypertension and Vascular Research Center, Wake Forest University School of Medicine
Application: Western blotting
Species + Tissue/Cell type: Rat dorsal medulla brain & cortax + hypothalamus extract
How many ug's of tissue/cell lysate run on the gel: 1. 40ug rat dorsal medulla brain extract 2. 20 ug rat cortax + hypothalamus mitochondria extract
Primary antibody dilution: 1:2500
Secondary antibody: Anti-Rabbit HRP
Secondary antibody dilution: 1:5000
How do Aviva's reagents play a role in your experimental goals?
To determine oxidative stress status in our animal models.
How would you rate this antibody on a scale from 1-5 (5=best) and why?
4- we see bands at expected MW about 24 kDa, however we do see some non-specific bands at higher MWs.
Would you use this antibody in future experiments?
Have you used another antibody which has worked in your application?
If the antibody works, do you plan to use it in future experiments or to publish your data? Why or why not?
Sample Description (please include 1) species type, and 2) cell/tissue type, and 3) how much protein loaded for each lane of your gel):
Rat brain: lane #1 is dorsal medulla tissue extract (40 ug) and lane # 2 is isolated mitochondria from rat brain cortex and hypothalamus pooled (20 ug).
How many different experimental trials were conducted using the antibody sample?
What type of experimental sample are you using and how did you preparing it?
"Lane # 1: homogenize brain dorsal medulla in 20mM potassium phosphate buffer, pH 7.0 with 1mM EGTA and Sigma protease cocktail inhibitor; and prepare samples in Laemmli buffer for WB
Lane # 2: isolate brain mitochondria (cortex and hypothalamus pooled) and prepare samples in Laemmli buffer for WB"
Primary used and dilution:
1: 2500 in 5% milk-TBST (0.05% Tween) for overnight at 4 degrees.
Secondary used and dilution:
1: 5000 anti-Rabbit HRP in 5% milk-TBST for 2 hrs at room temperature.
"Gels: (cat#161-1155, BioRad) Ready Gel Tris-HCl Gel, 10% resolving gel, 4% stacking gel, 10-well, 50 ul,
Running buffer: BioRad cat # 161-0732 : 1X Tris/Glycine/SDS: run gels for 70 min at 120 V
Transfer buffer: BioRad cat # 161-0734 : 1X Tris/Glycine with 20% methanol: Transfer for 1 hr at 100V
Blocking buffer: 5% milk in TBS-Tween (0.05%) for 1 hr at room temperature
Primary antibody: overnight at 4 degrees
Wash 3 times with TBST for 10 min per wash
Secondary antibody: 2 hrs room temperature
Wash 3 times wiht TBST for 10 min per wash
Develop bands with chemiluminescent substrate (Pierce: SuperSignal West Pico Chemiluminescent Substrate)"
What controls were used in your experiment? Please include your positive control:
Tissue (positive), mitochondria (negative).
How did you store the antibody after re-suspension?
In water, at -80 degrees.