Application
Western blot
Sample
Human Cell lysate - whole cell
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10%
Other product details
Dilution
1/500
Incubation time
1 hour(s) and 0 minute(s)
Secondary antibody
Name
Non-Abcam antibody was used: goat anti-rabbit biotinylated (Vector BA1000)
Host species: Goat
Clonality: Polyclonal
Conjugation: Biotin
Host species: Goat
Clonality: Polyclonal
Conjugation: Biotin
Detection
Detection method
DAB
Exposure
10 minute(s) and 0 second(s)
Bands
Specific: 60 and 110 kDa Non-specific: 80 and 125 kDa
Positive control
Jurkat whole cell lysate, 10 microl of lysate containing 20 millions cells/ml.
Negative control
We were hoping HeLa and THP-1 whole cell lysates, 10 microl of lysates containing 20 million cells/ml.
Additional data
Additional Notes
We are definitely picking something with this antibody but the size does not correspond to what the datasheet describe. According to the nucleotide sequence GPR15 should be 40-41kD, according to the datasheet 50kD and we detect a product at approximately 60 and 110kD. There are a couple of other bands (of higher MW) but these are also showing when the blot is probed with a rabbit IgG and are therefore non-specific.
Tha datasheet show an image of heart lysate being positive, but Clayton et al, (2001) JAM 159(5):1933-1939, who prepared their own GPR15 antibody, detected a product of 37kD which was not expressed in heart lysate.
Deng et al, (1997) who cloned GPR15 (BOB), did not detect BOB (GPR15) mRNA in the heart. Finally we have now performed RT-PCR on Jurkat, THP-1 and HeLa cell RNA and confirmed that these cell line do not express mRNA for GPR15.
Tha datasheet show an image of heart lysate being positive, but Clayton et al, (2001) JAM 159(5):1933-1939, who prepared their own GPR15 antibody, detected a product of 37kD which was not expressed in heart lysate.
Deng et al, (1997) who cloned GPR15 (BOB), did not detect BOB (GPR15) mRNA in the heart. Finally we have now performed RT-PCR on Jurkat, THP-1 and HeLa cell RNA and confirmed that these cell line do not express mRNA for GPR15.
Abcam response
Thank you for your Abreview. Your data is very convincing and clearly demonstrates cross reactivity by this antibody against two non-specific targets in your THP-1 cells in addition to our recommended HeLa and Jurkat control cells. We have not received complaints about this antibody before, so it may be that this particular Lot is to blame or the antibody may have been compromised during shipping. We hope that you have already received the free of charge replacement vial of an alternative product and it works properly.
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.
Dr. Caroline Schmutz
Verified customer
提交于 Dec 05 2005