Buy kit and follow instructions

Krishna, one of the things people love about SA-biotin binding is that it's so robust. We used to say "roar and pour." This is likely to work well for you.

Here are a few tips.

1. Most people think "chemical bond" when they read "conjugation." So you may inadvertently confuse or offend colleagues using the word "conjugation" to mean irreversible but non-covalent binding.

2. Purify your A & B components separately before combining. This may be challenging, depending on your ultimate purity requirements and the particular impurity fragments in your crude sample.

3. Assuming your fragments are soluble, you may perform your coupling reaction in a mild buffer, like PBS. (If your A & B are poorly soluble, you may include a little Tween or guanidine.) To minimize limiting diffusion via viscosity, stir the reaction, and keep the protein concentration below ~100 ug/mL. To help control microbial spoilage do it in the fridge, for 1h or overnight, and consider including a biostat, like NaN3.

4. Calculate concentration of A & B, and include them at 1:1 molar ratio for binding.

5. Depending on your final application, you may need to purify again after binding, but you probably can accomplish that via facile molecular sizing.

Respected John Carter

Thank you for your valuable tips and correcting the terminology too. As you rightly mentioned i already purified my biotin labelled protein A and also mSA2 fused Protein B separately and both are soluble too. i was just thinking about the efficient incubation temperature to be at room temperature or with fridge.

Thank you once again for your detailed response. I let you know my results soon

Thanking You

Apologies for the late answer. To confirm the conjugation Native -PAGE was run and this was the result

1. No band for Protein A (when i analysed i guess it could be because the pI of this protein A is ~12. I could get the clear band at the expected size when i run it in conventional SDS-PAGE)

2. No band for biotin labelled Protein A (b-ProtA)

3. mSA2 Protein B band was there but it was not resolved down the gel as that of the conventional SDS-PAGE (pI is ~5)

4. b-ProtA conj. with mSA2 Prot B: Band appearing higher than the Sl.no. 3 giving some hope there could be conjugation but it is faint

Now looking for some way to confirm my conjugation experiment.

I had a plan to do Westernblot with HRP streptavidin to confirm the binding of the biotin with protein A.

Additionally i tried to reverse the polarity and run since the pI is 12 but im not sure since the pI 12 protein conjugated with pI 5 protein

Thanking You