2 replies to this topic

[bdg25]

Hi all,

just a few technique questions.

 

1.) Do you sterile filter all reagents (TBST, blocking buffer, etc.) before use

2.) How long do you wash for each wash cycle? I've been washing 5 minutes each 3x for each wash step - is this excessive?

3.) When diluting antibodies, how do you ensure equal mixing? Do you pipette up and down? Do you vortex?

4.) How long do you block for? How long do you apply your antigen/sample/analyte? I've been doing two hours at RT. I'm using cell lysate and was wondering if I need to put a protease inhibitor in the solution if incubating for two hours.

 

 

Sorry for all of the questions,  I know some have been covered elsewhere but I'm finding a lot of conflicting information between different sources! 

[bob1]

The conditions you should use will depend on the antibody and antigen being detected.  However:

 

1) no, it shouldn't be necessary as it is easier and cheaper to make fresh stocks if needed.

2)antibody dependent - 3x5min is probably OK, but you could go as high as 6x5min.

3)Depends on volume.  I usually just invert the tube a few times.

4)Block 1/2 -1 hour. Antigen - antigen and antibody dependent, most will work in the 1-2hours at room temp.  Protease inhibitors are usually good things to add.

[bdg25]

Thanks Bob1 -- all about what I thought but I had been obsessively sterile filtering all reagents and it was really becoming a time sink and didn't make a whole lot of sense. Anyway, thanks for the help

ben