Application
Immunoprecipitation
Sample
Human Cell lysate - other (HELA)
Total protein in input
200 µg
Specification
HELA
Immuno-precipitation step
Protein G
Other product details
Incubation time
4 hour(s) and 0 minute(s) · Temperature: 4°C
Western Blot antibody
Name
Abcam antibody:Anti-Stromal interaction molecule 1 antibody
Dilution
1/1000
Additional data
Additional Notes
Could you please provide the following information:
1.Could you confirm that the antibody incubation time for IP was 1h? Antibody was incubated with beads for just 1 h. (Time changed from 1h to 4h).
2.How much antibody did you use for 200ug of HeLa cells for IP?
10ul of undiluted antibody+30 ul bead matrix-incubated for 1h-mixed with 200ug of cell lysates (another 4h incubation) for IP
3.What IP buffer did you use? 1% CHAPS lysis buffer
4.how did you carry out elution? Beads in loading dye heated at 55 deg for 10 mins for elution
5.How many times did you carry out IP? Twice, so far
6.Looking at the image- the beads only control has high background- could you explain this?
The beads do not have background. Bands at 50 and 25KD are the heavy and light chains of antibody. Since its a heavy exposure, we see nonspecific faint bands in the lysates which happen to be around 50 and 36 KD.
7.Were in the image is the correct band? STIM1 is a 77 KD protein. So, the strongest band above 64 KD marker is STIM1
8.Basis of rating the antibody suitability excellent for IP? Because the antibody pulled down a good fraction of endogenous STIM1
In WB:
1.how long was the antibody incubation time? 16h
2.How much sample did you load on the gel? 50ug
3.what buffer was the antibody diluted in? TBST
4.did you carry out a no primary control for the secondary?
Never needed to run secondary controls. As I have these samples blotted for other antibodies with similar and different secondaries, giving specific bands
1.Could you confirm that the antibody incubation time for IP was 1h? Antibody was incubated with beads for just 1 h. (Time changed from 1h to 4h).
2.How much antibody did you use for 200ug of HeLa cells for IP?
10ul of undiluted antibody+30 ul bead matrix-incubated for 1h-mixed with 200ug of cell lysates (another 4h incubation) for IP
3.What IP buffer did you use? 1% CHAPS lysis buffer
4.how did you carry out elution? Beads in loading dye heated at 55 deg for 10 mins for elution
5.How many times did you carry out IP? Twice, so far
6.Looking at the image- the beads only control has high background- could you explain this?
The beads do not have background. Bands at 50 and 25KD are the heavy and light chains of antibody. Since its a heavy exposure, we see nonspecific faint bands in the lysates which happen to be around 50 and 36 KD.
7.Were in the image is the correct band? STIM1 is a 77 KD protein. So, the strongest band above 64 KD marker is STIM1
8.Basis of rating the antibody suitability excellent for IP? Because the antibody pulled down a good fraction of endogenous STIM1
In WB:
1.how long was the antibody incubation time? 16h
2.How much sample did you load on the gel? 50ug
3.what buffer was the antibody diluted in? TBST
4.did you carry out a no primary control for the secondary?
Never needed to run secondary controls. As I have these samples blotted for other antibodies with similar and different secondaries, giving specific bands
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提交于 Sep 28 2010