Question

Here are the results obtained by Western type immunoblotting on cell signaling proteins. WB represent 4 experimental conditions (time 0, 5, 15 and 30 minutes of stimulation with a ligand on total cellular extracts)

Question 6. Why use an antibody that recognizes the phosphorylated form (p-ERK) and an antibody that recognizes the ERK proteins? Why are there 2 bands recognized by these antibodies? (8 lines maximum)

Question 7. How do we use the WB results to obtain a histogram result? (3 lines maximum)

Question 8. Please give me the main steps (diagram or protocol by numbering) of the migration protocol on acrylamide gel followed by WB using as an example the visualization of p-ERK (1 page max

Western Blot (WB) ou immunobuvardage de type Western Voici des résultats obtenus par immunobuvardage de type Western sur des protéines de signalisation cellulaire. Les WB représentent 4 conditions expérimentales (temps 0, 5, 15 et 30 minutes de stimulation avec un ligand sur des extraits totales cellulaires) I Question 6. Pourquoi utiliser un anticorps qui reconnait la forme phosphorylée (p-ERK) et un anticorps qui reconnait les protéines ERK? Pourquoi y a-t-il 2 bandes reconnues par ces anticorps? (8 lignes maximum)

thats the only information i have about the Western Blot.the 4 conditions (times)

Answer 1

Question 6. ERK antibody detects all activated and non-activated ERKs in the cell. But p-ERK antibodies detect only the phosphorylated ERK present in the cell. There are two isoforms of ERK p44 (44kDa) and p42 (42kDa), for which there are two bands in the blot.

Question 7. The band intensity in the western blot can be measured in the software like ImageJ, GelQuant, etc. Those intensities can be plotted in a graph normalized with the loading control. This way you can get the histogram.

Question 8. The steps are as follows

Prepare the sample so that phosphorylation of protein is not hampered

Run in a SDS-PAGE

Transfer the separated proteins in the gel according to molecular weight to a PVDF membrane.

Do blocking of the PVDF membrane with Bovine Serum Albumin.

Incubate with primary antibody

Incubate with secondary antibody

Develop with a chemiluminescent substrate.

Detect the bands and interpret the result.