Protein Loading and Western Blot - Western Blot (Dec/24/2008 )
Hello,
I was wondering if anyone knew what determines the amount of protein in ug that should be loaded for proper detection in a Western.
I was going to load 50ug/lane but after quantifying the proteins extracted from primary culture cortical neurons I found that my samples are way too dilute and I don't have enough left to load this amount.
I am thinking about loading 35ug instead but am worried that the proteins I will be probing for may not be detected.
Will loading 35ug be a problem and is there anyway I can possibly concentrate my samples magically?
Thanks very much for all your help and Merry Christmas,
llama
between 15ug-25ug gives me good results.
Thanks very much for your help.
Take care!
This very much depends on the protein you are hoping to identify and the antibody you are going to use.
If the endogenous protein is expressed at low levels, you are going to need to run more extract in order to detect it. Some proteins are cell-cycle regulated or expression is greatly altered by specific stimulation or conditions so you'll get different results from synchronized cells or cells that have been stimulated/treated. For example, I used to stimulate cells with insulin and look at the phosphorylation status of specific kinases. Each protein is different and you will have to work out the best for conditions for your specific protein.
Another major factor is the antibody being used in the western. Some antibodies are highly sensitive and have a high affinity for the protein of interest. This allows you to run much less lysate and still have good detection. On the other hand, some antibodies just suck and are barely worth working with. Again, it's all about your specific reagent. All I can add here is that if you are getting no signal (or very weak) it may be worth doing a third probe with protein A-HRP. This will bind to both the primary and secondary antibodies and increase any signal.
If the endogenous protein is expressed at low levels, you are going to need to run more extract in order to detect it. Some proteins are cell-cycle regulated or expression is greatly altered by specific stimulation or conditions so you'll get different results from synchronized cells or cells that have been stimulated/treated. For example, I used to stimulate cells with insulin and look at the phosphorylation status of specific kinases. Each protein is different and you will have to work out the best for conditions for your specific protein.
Another major factor is the antibody being used in the western. Some antibodies are highly sensitive and have a high affinity for the protein of interest. This allows you to run much less lysate and still have good detection. On the other hand, some antibodies just suck and are barely worth working with. Again, it's all about your specific reagent. All I can add here is that if you are getting no signal (or very weak) it may be worth doing a third probe with protein A-HRP. This will bind to both the primary and secondary antibodies and increase any signal.
Hello rkay447,
I was thinking of doing a test western and loading different amounts of lysate [ 10ug, 20ug, 35ug, and 50ug] to see which one gives me the best signal with the antibodies that I will be using. I have been searching the literature to see what other groups have done when probing for my protein but that has been proven to be quite time consuming.
Thanks very much for all your help. I really appreciate the time you took to respond to my question.
Take care,
llama
If the endogenous protein is expressed at low levels, you are going to need to run more extract in order to detect it. Some proteins are cell-cycle regulated or expression is greatly altered by specific stimulation or conditions so you'll get different results from synchronized cells or cells that have been stimulated/treated. For example, I used to stimulate cells with insulin and look at the phosphorylation status of specific kinases. Each protein is different and you will have to work out the best for conditions for your specific protein.
Another major factor is the antibody being used in the western. Some antibodies are highly sensitive and have a high affinity for the protein of interest. This allows you to run much less lysate and still have good detection. On the other hand, some antibodies just suck and are barely worth working with. Again, it's all about your specific reagent. All I can add here is that if you are getting no signal (or very weak) it may be worth doing a third probe with protein A-HRP. This will bind to both the primary and secondary antibodies and increase any signal.
Hello rkay447,
I was thinking of doing a test western and loading different amounts of lysate [ 10ug, 20ug, 35ug, and 50ug] to see which one gives me the best signal with the antibodies that I will be using. I have been searching the literature to see what other groups have done when probing for my protein but that has been proven to be quite time consuming.
Thanks very much for all your help. I really appreciate the time you took to respond to my question.
Take care,
llama
In this case I would suggest you to load less amount of protein, say 1ug to 20ug, or you just load 5ug of your protein and use different primary Ab dilution. Of course I assumed that your Ab has high affinity. Where did your Ab come from? homebrew or purchased?