Western blot abreview for Anti-GPCR GPR43 antibody - Cytoplasmic domain

Poor
Abreviews
Application
Western blot
Sample
Human Cell lysate - whole cell (Cell line (HT29))
Gel Running Conditions
Reduced Denaturing (10%)
Loading amount
20 µg
Specification
Cell line (HT29)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 24°C

Other product details

Incubation time
14 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: 3% Skim milk /TBS-T
Dilution
1/1000

Secondary antibody

Dilution
1/5000
Name
Non-Abcam antibody was used: Donkey anti-Rabbit IgG (H+L) Cross Adsorbed Second
Host species: Donkey
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase

Detection

Detection method
ECL+,
Negative control
HCT116 cell is known to be deficient with GPR43. We confirmed it by PCR. We made the whole cell lysate with RIPA buffer and used as positive control.
Exposure
20 minute(s) and 0 second(s)
Positive control
HT29 cell is previously reported to express GPR43 (Int. J. Cancer, 847-856 2011). We confirmed it by PCR. We made the whole cell lysate with RIPA buffer and used as positive control.

Additional data

Additional Notes
We used regular ECL reagent first (Chemi-Lumi one L, Nacalai #07880-70) which gave us bands for b-actin but not for GPR43 (with no non specific bands). We further tried it with highly sensitive ECL reagent (ImmunoStar LD, Wako #269-69901) which showed many non specific bands but not the size of GPR43. We attached a GIF file showing this (Maker, Low range Nacalai #28941-75). Left lane is HT29, and right lane is HCT116.
Abcam response
Thank you for your valuable feedback and for participating in our Abtrial testing program. We are sorry to hear that this antibody has not given you good results in Western Blot. This antibody has not been tested with this application before, and it is possible that it may not be suitable for use.

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Submitted Apr 22 2016

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