Normalization of western blot bands ll Statistical analysis ll xgene and proteinX

Normalization of western blot bands ll Statistical analysis ll Xgene and proteinX

[@sangeethamsbd]

Hi, on western blot visually 5 and 10 ug protein band is more intense compared to control but after normalization on the graph it shows control is higher amount compared to two other bands, why?

[@xgeneproteinx2889]

Normalization value not only depends on intensity of the protein bands of test group. It also depends on intensity of the loading control. If you notice, you can see intensity of GAPDH (loading control) of corresponding control group is very low. This is why normalization value is less in the control group.

[@sangeethamsbd]

@@xgeneproteinx2889 Thanks for the reply. Can you please help me out with a query, shall i normalize western blot bands with total protein gel stained with Coomassie?

[@xgeneproteinx2889]

@sangeethamsbd Usually not, how do you get loading control in the SDS PAGE?

[@SabraFatima-p2n]

Thank you. Highly appreciable that you took your time and responded to our queries. It was really helpful. 😊🌸

[@xgeneproteinx2889]

Thanks for your appreciation.

[@Prem25119]

This video is so helpfull. Thank you so so much

[@xgeneproteinx2889]

Thanks for your comment.

[@FarihaTasnim-ro4fl]

So thankful for this video. If my bands have some background how do I remove it? and one more question, if I have 3 drugs group and 12 samples total, what would be the control here?

[@xgeneproteinx2889]

Thanks for watching. 1. There are several points should be considered to get rid off background noise. I'll make a video on it soon. 2. Usually, Should have an untreated group as control. Better to add a control group to do statistical analyses.

[@xgeneproteinx2889]

Video on 'western blot troubleshooting tips' uploaded. You can check it out. Hope it will be helpful.

[@krystalkaleb5823]

Your explanations are best

[@xgeneproteinx2889]

Glad it helped.