I need to perform western blotting but i have no practical experienced yet.
Please kindly suggest simple and accurate procedure for Western Blot.
And what dye/stain will is reversible for protein detection suitable?
Thank you,
Dear Mathipi,
The simple steps are, 1.Sample preparation for WB. 2. Loading of sample in SDS-PAGE (percentage depends on MW of the protein of interest) and run. 3. Transfer the proteins into membrane. 4. Blocking of the membrane using 1% BSA or 5% skimmed milk. 5. Incubation with primary antibody. 6. washing in PBST/TBST. 7. incubation with secondary Ab (conjugated with HRP). 8. Washing. 9. Detection of fluorescence. 10.Quantitative densitometric analysis of bands using software. For every details and better understanding, it is better to contact with the person who is performing WB. The answers will give you a theoretical view only. Kindly go through the attached files which will give you an insight view of WB techniques.
Hope it will help.
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