Application
CLIP
Sample
Human Cell lysate - whole cell (HEK 293T)
Specification
HEK 293T
Type
iCLIP
Irradiation Energy Level (mJ/cm2): 150
Wavelength (nm): 254
Irradiation Energy Level (mJ/cm2): 150
Wavelength (nm): 254
Positive control
hnRNP C antibody (sc-15386) bound proetin A dynabeads, 0.1 mg/ml (antibody/beads)
Negative control
Protein A dynabeads without antibody bound
Immuno-precipitation step
Other - Protein A dynabeads
Other product details
Incubation time
16 hour(s) and 0 minute(s) · Temperature: 4°C
Additional data
Additional Notes
The samples were treated with high RNase, the positive hnRNP C control displays strong signal at 40 KDa, as expected. The RBM14 sample showed noise obscuring the region >64 KDa. The efficiency of IP was assessed by performing western blot on the lysate before (HEK 293T) and after IP with or without ab70636 antibody (RBM14 HEK or no antibody HEK 293 resp.) Strong bands were detected at 64 KDa, however little to no depletion was observed after IP.
Top image:
Lane 1: hnRNP C iCLIP in HEK 293T (positive control)
Lane 2: RBM14 iCLIP in HEK 293T
Autoradiogram exposure – 1 hour at -80C
Western:
Primary: 1:5000 ab70636 16 hours at 4C
Secondary: 1/2000 goat anti-rabbit HRP 1 hour at room temperature
exposure 40min RT
The amount of cell lysate was quantified by measuring the concentration of nucleic acid (absorbance at 260 nm) and using ~100ug/ml.
This is added to the protein A dynabeads with 0.1 mg/ml antibody bound, only 0.1ml beads are used so total antibody (assuming 100% binding to dynabeads) 10ug.
Top image:
Lane 1: hnRNP C iCLIP in HEK 293T (positive control)
Lane 2: RBM14 iCLIP in HEK 293T
Autoradiogram exposure – 1 hour at -80C
Western:
Primary: 1:5000 ab70636 16 hours at 4C
Secondary: 1/2000 goat anti-rabbit HRP 1 hour at room temperature
exposure 40min RT
The amount of cell lysate was quantified by measuring the concentration of nucleic acid (absorbance at 260 nm) and using ~100ug/ml.
This is added to the protein A dynabeads with 0.1 mg/ml antibody bound, only 0.1ml beads are used so total antibody (assuming 100% binding to dynabeads) 10ug.
Abcam response
We appreciate collaborator's feedback on ab70636 suitability for iCLIP.
IP conditions might need to be optimized further.
We welcome any feedback our customer's have on this antibody.
IP conditions might need to be optimized further.
We welcome any feedback our customer's have on this antibody.
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 Jun 02 2011