We used to use 5% milk to block NC membrane and incubate the membrane in Primary Antibody (1% Milk and TBST) and we used to reuse the Primary Ab (Storing in -20C). That time it worked nice.
Now, we are using Li-cor Intercept Blocking buffer and see that after the first use, the primary Ab is not working. We are diluting the Primary Ab in 1% Milk and TBST, still, we are getting the same result.
We called Cell signaling (For Antibody) and Li-cor Bio, both told they have no data, also, they told they don't recommendation using the Primary Ab to reuse.
Does anyone face same kind of issue?
I have a question for you. After you reconstitute your antibody from cell signaling into either licor buffer or milk and use it for western blots, are you then placing the diluted antibody back into the freezer? If you are exposing your diluted antibody to multiple freeze thaws that could be a potential source of your decaying signal.
most, if not all, antibody sotcks from cell signaling are stored at -20 as a stock because it has glycerol to prevent freezing. Once you have reconstituted your primary antibody into licor buffer or milk, keep it at 4 degrees.
Also, are you looking at a phosphorylated antibody? Those can be tricky and susceptible to degradation.
I have used both the original licor buffer as well as the "new" intercept licor buffer and have not had any problems. I add sodium azide to a final concentration of 0.02% in the diluted antibody and store it at 4 degrees for up to a month. Within that time period I have used the antibody multiple times with no problems.
Brandon A Kemp I keep the Primary Ab into -20C after using it for Western. Earlier, we used to keep them (Same Ab) into -20C and was giving good result (When we were using ECL set up). I don't know if there is any interaction between Li-cor buffer and Ab.
I have a question for you, do you mix sodium azide while diluting the Primary Ab for the first time or after the first time use?
Anyhow, thanks for providing a valuable suggestion.
Hey, no problem at all. I would advise you not to put your diluted/reconstituted antibody back in -20 after using it, unless you have added a percentage of glycerol. You want to avoid freeze thaws with antibodies. I would store it at 4 degrees after you defrost the stock antibody from cell signaling.
In terms of sodium azide, you can add it to the primary antibody at any time. It does not interfere with binding. Usually, I add it after the first use. If you are performing chemi detection with ECL, remember, do NOT add sodium azide to any solution that contains the secondary antibody. This prevents the light reaction from occurring and you will see nothing. I am not sure if this pertains to fluorescence imaging, but you would not need to add sodium azide to any secondary solution since that is not kept and stored. It is a one time use.
Brandon A Kemp
Thanks for providing a clear idea.
I want to ask this, please let me know if you can unveil this doubt:
We used to keep them (Same Ab from cell signaling) into -20C. We reused them couple of time and they was giving good results (When we were using ECL set up, not using the Li-Cor buffer. We were using 5% milk for blocking-->1% Milk and TBST for Primary Ab and Secondary Ab dilution).
After started using the Li-cor buffer, we have started facing this issue. I don't know if there is any interaction between Li-cor buffer and Ab.
Anyhow, I will use your technique to store my Ab. Thanks once again.
I am not sure why you are having problems with your fluorescence detection. One thing that crossed my mind is, did you add tween to your licor solution containing the antibodies? When you use milk in TBST it has tween, but when you use licor buffer you need to add it.
In general, fluorescent detection is not as sensitive in my opinion compared to ECL detection because ECL detection amplifies the signal with multiple tags on the secondary antibody. If the milk works, which your concentrations/percentages are fine, why are you bothering with the licor buffer?
If you are committed to performing fluorescent detection, you can still use milk for blocking and antibody dilution. I have done this all of the time and it works just fine. Also, I use blotto non fat dry milk from santa cruz.
good luck
Brandon A Kemp Thanks for your reply.
Yes, I add 0.2% Tween20 to blocking buffer as per Li-Cor Protocol. I am trying to find out the reason why Ab is degrading.
Is it fine if I can send you one-to-one message? I want to know about your protocol of blocking with 5% milk and diluting the Primary Ab in 1% Milk TBST solution.
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