Nf Isaab Lab Report
Lab report NF-κB
1. Aim
The aim with this lab was through using ELISA-based technique learn how IL-1 and LPS activate the NF-κB signaling pathway in insulin-producing cells and see if the activation can be prevented through inhibition of proteasomal activity. In addition the purpose of this lab was to detect whether NF-κB activation leads to induction of the iNOS enzyme or not.
2. Background
NF-κB is a transcription factor that in its inactive form is bound to IκB, which is a cytoplasmic inhibitory protein. NF-κB itself is composed of two subunits, p65 and p50 and is a heterodimer. The bacterial protein LPS and the receptor for the cytokine IL-1 can activate the NF-κB signaling pathway which induces phosphorylation and ubiquitination of IκB. This will make a release of NF-κB through proteasomal degradation.
3. Methods
3.1 Incubation of cells with different test substances …show more content…
The cell cultures were placed in four wells so it would be possible to determine if a specific substance can activate NF-κB. Well #1 was used as a control with no additions of substances. In well #3 the proteasome inhibitor TLCK was added and incubated for five minutes, thereafter there was an additional five minutes wait before the addition of other substances to the wells to make sure the inhibitor fully diffused into the cells. Following, in well #2 the bacterial protein LPS was added and in both well #3 and #4 cytokine IL-1 before the whole cell culture plate was incubated for 15
1. Aim
The aim with this lab was through using ELISA-based technique learn how IL-1 and LPS activate the NF-κB signaling pathway in insulin-producing cells and see if the activation can be prevented through inhibition of proteasomal activity. In addition the purpose of this lab was to detect whether NF-κB activation leads to induction of the iNOS enzyme or not.
2. Background
NF-κB is a transcription factor that in its inactive form is bound to IκB, which is a cytoplasmic inhibitory protein. NF-κB itself is composed of two subunits, p65 and p50 and is a heterodimer. The bacterial protein LPS and the receptor for the cytokine IL-1 can activate the NF-κB signaling pathway which induces phosphorylation and ubiquitination of IκB. This will make a release of NF-κB through proteasomal degradation.
3. Methods
3.1 Incubation of cells with different test substances …show more content…
The cell cultures were placed in four wells so it would be possible to determine if a specific substance can activate NF-κB. Well #1 was used as a control with no additions of substances. In well #3 the proteasome inhibitor TLCK was added and incubated for five minutes, thereafter there was an additional five minutes wait before the addition of other substances to the wells to make sure the inhibitor fully diffused into the cells. Following, in well #2 the bacterial protein LPS was added and in both well #3 and #4 cytokine IL-1 before the whole cell culture plate was incubated for 15