Use of FCS for blocking

[Guest thomson]

Hi, 

Regarding blocking:

1. Is it sufficient to block the PVDF membrane with 200uL of culture media(10%FCS) incubate at RT for 30min?

2. Is it necessary to add 0.5% FCS in PBS as diluent for detection antibody and Streptavidin-AP? Is it okay with just pure PBS?

Also,

3. A few wells tend to have a darker background than others (and it's consistently occurs the same position added with a specific peptide pool!).The number of spots were not much so I wonder if something peculiar about the particular tube of pool that leads to such phenomenon. Impurities in the peptide etc?

 

Thanks!

Thomson

[Christian@mabtech.com]

Hi Thomson!

 

1. Yes, it is sufficient. 

2. The 0.5% FCS is added in order to "protect" the detection mab from getting stuck to the sides of the tube. In pure PBS plastic has a certain absorption. The 0.5% FCS stops this from being a factor. However, using pure PBS works. Certain plastics can absorb more detection mab and if you are using only 0,1 ug/ml of detection mab, protecting that amount becomes more important compared to a detection concentration of 1ug/ml. 

3. We know from experience that some peptides bind to the PVDF membrane and causes the detection mab to smudge more. We dont know why exactly, but I have heard theories of crystallization that remains even after the cells are washed away. Another possibility is that certain peptides does "something" to the cells, which in turn release factors to the membrane that causes the detection mab to stick more. Can be well exist other reasons. 

When this happens you ususally can still make out the spots against the slighly darker membrane. With a reader like Mabtech IRIS this is easy to compensate for using the RAWspot algorithm. 

[Guest thomson]

Thanks for the interesting information!

One last question, I've read in a thread where you recommended pure PBS to stop substrate development. 

Is there a reason not to use dh20 or tap water? Our lab simply rinse the plate with tap water. Would that be an issue?

[Christian@mabtech.com]

6 minutes ago, Guest thomson said:

Thanks for the interesting information!

One last question, I've read in a thread where you recommended pure PBS to stop substrate development. 

Is there a reason not to use dh20 or tap water? Our lab simply rinse the plate with tap water. Would that be an issue?

For ELISpot detected with ALP you can use tap water, PBS or dh20. Does not matter. 

However, for HRP spots we know from experience that certain water can cause the spots to turn yellow. As a result, I recommend PBS with a controlled pH for turning off HRP plates.