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How Can I Control My Loading?

How Can I Control My Loading? - Western Blot Forum

How Can I Control My Loading? - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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Old 07-22-2011, 05:04 AM
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Default How Can I Control My Loading?



Hi,
I'm posting something for the first time in this forum though I have learned a lot from others' posts. I am trying to understand whether chemoattractants and/or leukocyte adhesion to endothelial cells phosphorylate my target protein in endothelial cells. After optimizing several things, I have got my desired band. But the problem is I don't know how I can control my loading. I have different treatment groups (chemoattractants and stimulated leukocytes esp. neutrophil). In chemoattract groups I have only endothelial cells and I can use beta actin or other internal controls but in stimulated leukocyte treated groups there are two types of cells endothelial cells plus adherent leukocytes. So, there is more beta actin and total protein in leukocyte treated groups. How can I compare chemoattractant and leukocyte treated groups. Only thing I can control and in fact I am controlling is the number of endothelial cells. Neutrophils that I am using are deficient of my target protein. Now I am feeling helpless. Any suggestions? Thanks.
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Old 07-25-2011, 04:42 AM
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Default Re: How Can I Control My Loading?

To control loading of western blots, simply use a pipette that releases slowly - sometimes the old one.

Also make sure you use steady slow pressure on the release.

You can load well if you use a well prepared sample loading lammeli buffer which has good amounts of sucrose in it
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