How to choose secondary antibody?

[newbie17425]

Hi,

I wanted to know how should I decide which secondary antibody should I go for?
For primary, I used an anti-rabbit. I know that I have to use a secondary anti-rabbit, so I used a goat anti-rabbit. But, how do I decide if I should choose Alexa Fluor 488 or Alexa Fluor 555 goat anti rabbit?

Thanks.

[mdfenko]

it depends on the detection system you use.

what is the wavelength of your laser?

if dual lasers then your preference. if you are double staining then use the other one.

you probably want to use 488.

[newbie17425]

it depends on the detection system you use.

what is the wavelength of your laser?

if dual lasers then your preference. if you are double staining then use the other one.

you probably want to use 488.

Is there any difference between the former and the latter? I have both the detection system. I saw in different papers that, one uses 488 and the other uses 555 for the same primary antibody, therefore, I wanted to know if there is any difference? Will both of them detect my protein?

[mdfenko]

the primary antibody will determine if you can detect your protein.

the secondary determines the method for detection. you are looking at fluorescence detection. alexa488 is probably used more than other forms.

but, which form you use depends on how you will use them (double staining, etc) and availability (maybe even price).