Application
Western blot
Loading amount
50 µg
Gel Running Conditions
Reduced Denaturing (8% resolution gel)
Sample
Human Cell lysate - whole cell (hepatocellular carcinoma)
Specification
hepatocellular carcinoma
Treatment
Inhibition of one gene's expression using lentiviral shRNA
Blocking step
Milk as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C
Other product details
Incubation time
12 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 5% non-fat milk in TBST(0.1% Tween 20)
Dilution
1/1500
Secondary antibody
Name
Non-Abcam antibody was used: ECL Anti-rabbit IgG, Horseradish Peroxidase linked
Host species: Rabbit
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Host species: Rabbit
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Dilution
1/3000
Detection
Detection method
ECL
Bands
Specific: 60-65 kDa Non-specific: 55, 75 kDa
Exposure
2 minute(s) and 0 second(s)
Additional data
Additional Notes
I recently found inhibition of one gene which is overexpressed in hepatocellular carcinoma lead to decrease in TRF1 mRNA level (see Fig.A). Then I bought a TRF1 ab (ab1423) from Abcam to test if the protein level was also decreased. However, my WB results demonstrated two non specific bands at around 55KDa and 75KDa (see Fig.B and C, two different time of exposure respectively) in all of the samples.Only a weak band at around 60-65 KDa was developed as the datasheet claimed. The most important problem is no difference between the cells expressing control shRNA and targeting gene shRNA.
Abcam response
Our Scientific Support team is currently investigating this negative Abreview, and we will resolve the issue in accordance with our Abpromise.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
Abcam user community
Verified customer
提交于 Jul 16 2013