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 (sc-15386) bound protein 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
iCLIP with E18 mouse brain whole cell lysate was also performed under the same conditions and is shown in the image provided.
The samples were treated with high RNase, however the signal in iCLIP was at the wrong molecular weight in the HEK 293T lysate and the E18 brain signal was obscured by noise. The efficiency of IP was assessed by performing western blot on the lysates before (HEK 293T/E18 brain) and after IP with or without ab70455 antibody (ab70455 HEK/E18 brain or no antibody HEK 293/E18 resp.) Partial depletion can be observed in the HEK 293T lysate, but no depletion was observed in the E18 brain lysate.
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: positive control HEK 293T- hnRNP C antibody
Lane 2: positive control E18 brain- hnRNP C antibody
Lane 3: DDX6 HEK 293T- ab40684
Lane 4: DDX6 E18 brain- ab40684
Western:
Primary: 1/2500 DDX6 ab70455, 16h 4deg
Secondary: 1/2000 goat anti-rabbit HRP 1h RT
The western shows a comparison between the lysate before and after IP with ab40684, ab70455 or protein A dynabeads without antibody bound (as indicated), therefore we are looking to see depletion of the protein from the lysate.
The samples were treated with high RNase, however the signal in iCLIP was at the wrong molecular weight in the HEK 293T lysate and the E18 brain signal was obscured by noise. The efficiency of IP was assessed by performing western blot on the lysates before (HEK 293T/E18 brain) and after IP with or without ab70455 antibody (ab70455 HEK/E18 brain or no antibody HEK 293/E18 resp.) Partial depletion can be observed in the HEK 293T lysate, but no depletion was observed in the E18 brain lysate.
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: positive control HEK 293T- hnRNP C antibody
Lane 2: positive control E18 brain- hnRNP C antibody
Lane 3: DDX6 HEK 293T- ab40684
Lane 4: DDX6 E18 brain- ab40684
Western:
Primary: 1/2500 DDX6 ab70455, 16h 4deg
Secondary: 1/2000 goat anti-rabbit HRP 1h RT
The western shows a comparison between the lysate before and after IP with ab40684, ab70455 or protein A dynabeads without antibody bound (as indicated), therefore we are looking to see depletion of the protein from the lysate.
Abcam response
We appreciate collaborator's feedback on CLIP and are sorry to hear that the results were not positive.
However, the results show that Mouse species is suitable for this antibody.
However, the results show that Mouse species is suitable for 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.
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投稿 Jun 02 2011