IP washing step

[david1259]

I looked for several books and protocols, however I couldn't figure out.
I have high background in IP and there is not even candidate band either.
I didn't know the result is right because I don't know how to do IP very well.


In the protocols, there are just simple words..
"Add lysis buffer 0.5~1ml and centrifuge 2500g and discard sup. Do same washing more than 4 times.

These are what I would like to know

1. Does it matter to use lysis buffer or lysis buffer with inhibitor during washinhg step?

2. do I have to vortex or shake up and down everytime after adding lysis buffer?

3. If I added 1ml buffer, do I have to take out almost 1ml or a lot less after centrifugation?

4. What is efficient way to take out buffer with not touching bead?

Please, help me ... I am stucking in this IP problem for several weeks.

Thank you,

[danfive]

So you are washing beads with 'lysis buffer'? If you can post the buffer recipe.

For your other questions:
1. protease inhibitor doesn't matter in wash step, they are usually used in the initial capture incubation period.
2. If you mean wash buffer, yes mix by gentle vortex or inverting tubes.
3. If washing, its best to remove as much of the wash buffer as possible, without picking up beads.
4. Simple filter spin columns that retain the beads and allow wash to flow through.

[danfive]

Originally Posted by david1259

I looked for several books and protocols, however I couldn't figure out.
I have high background in IP and there is not even candidate band either.
I didn't know the result is right because I don't know how to do IP very well.

Thank you,

Maybe its time to try Pierce Seize X Protein G Immunoprecipitation Kit. I used it multilple times and highly recommend it. Here is a product manual maybe the instructions will help you ---> http://www.piercenet.com/files/0936as4.pdf

[david1259]

My lysis buffer is RIPA

50 mM Tris-HCl , pH 7.4
150 mM NaCl
1 mM EDTA
1% Triton x-100
1% Sodium deoxycholate

Add protease inhibitor cocktail tablet, NaF and NaVO4 before use
0.1% SDS

[danfive]

Originally Posted by david1259

My lysis buffer is RIPA

50 mM Tris-HCl , pH 7.4
150 mM NaCl
1 mM EDTA
1% Triton x-100
1% Sodium deoxycholate

Add protease inhibitor cocktail tablet, NaF and NaVO4 before use
0.1% SDS

Please clarify if the Ripa lysis buffer is being used as wash buffer also. As this can be a very big problem.
The foremost problem is the SDS it will dissociate the antigen-antibody complex, next the salt concentration from Tris, NaCl and NaDeoxycholate can also dissociate the capture complex. Try PBS-Tween for a wash.

[david1259]

Since I use specific beads, I couldn't use IP kit, I placed an order for spin cup not but IP kit. For the spin cup, putting the resin-sample, cenrifuge and elute sample. Am I right? Also, I am going to use PBS-tween for washing buffer rather than RIPA. Thank you for all your help. Have a nice Holiday.

[danfive]

Originally Posted by david1259

Since I use specific beads, I couldn't use IP kit, I placed an order for spin cup not but IP kit. For the spin cup, putting the resin-sample, cenrifuge and elute sample. Am I right? Also, I am going to use PBS-tween for washing buffer rather than RIPA. Thank you for all your help. Have a nice Holiday.

You can add the resin-beads + captured protein to the spin cup and perform all your wash steps and centrifuge to get rid of the wash flow-through, your captured protein will stay in the antigen-antibody complex in the cup, until you add an elution buffer (high salt or low pH buffer). Also most protocols recommend something like 8-10 wash steps at most, but I found that in order to get rid of a strong albumin band co-eluting with my immunocaptured protein I had to boost the wash steps to 30, and it worked incredibly well.

[bobbylee]

You can try washing with PBS + 2% Triton X-100. Add wash buffer, vortex then centrifuge at max speed in a table-top sized centrifuge for ~1min at 4 degrees- repeat another 2x. After last wash remove as much buffer as you can without disturbing beads. Another way to remove buffer is to set up a vacuum (using standard vacuum on a lab bench) and attach a syringe and fine needle. Just make sure the vacuum goes through a beaker of some description to dispose of the liquid if doing this way.

[danfive]

Originally Posted by bobbylee

You can try washing with PBS + 2% Triton X-100. Add wash buffer, vortex then centrifuge at max speed in a table-top sized centrifuge for ~1min at 4 degrees- repeat another 2x. After last wash remove as much buffer as you can without disturbing beads. Another way to remove buffer is to set up a vacuum (using standard vacuum on a lab bench) and attach a syringe and fine needle. Just make sure the vacuum goes through a beaker of some description to dispose of the liquid if doing this way.

?????? For IP you're going to need more than three wash steps.

[bobbylee]

Have never had a problem. Get a nice band with no background/non-specific binding when running a western.