Assaying H1 kinase in extracts (Stukenberg lab) and Immunoprecipitation (Stukenberg lab): Difference between pages

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Histone H1 Kinase Assay from Xenopus Extracts
IP Protocol
This protocol utilizes the coupling of Antibody to Protein A Coated beads using DMP.  This allows for elution of bound proteins and reuse of beads, if desired. 


Materials:


Purpose:  Test whether the H1 kinase Cdc2-CcyclinB is still active in an extract. This is a readout for the extracts mitotic state. If activity is high, the extract is mitotic, if it is low, the extract has exited mitosis and is in interphase. These are normally done in our lab as a readout for the spindle checkpoint. An extract containing sufficient sperm and nocodazole will maintain a mitotic state after being released from CSF, by the addition of calcium.  
Bio-Rad Protein A Support
 
Control IgG (depending on antibody source; rabbit, mouse, etc..)
 
PBSt (PBS + 0.1% Tween20)
 
0.2 M Sodium Borate pH 9.0
 
0.1M Glycine, pH 2.5
 
Trichloroacetic Acid (TCA)
 
1% Deoxycholic acid (DOC)
   
   
Background:  Snap freeze in liquid nitrogen 1µl samples of the extract to be analyzed.  Store at -80°C until you are ready to perform the kinase assay.
Prepare a mixture of the following reagents, making enough for all kinase assays to be performed at one time.
   
   
Methods
1. Remove an appropriate amount of Protein A beads and wash 3 times with PBSt. You will need a minimum of 2 tubes of beads at this point, one for you’re your antibody of interest and one for control IgG.
2. Assuming you are using 10-100 µl of beads, add PBSt and antibody to a final volume of ~500µl. 
3. Mix the antibody/bead solution in the cold room for 1 hour to overnight.
4. Wash the beads 3 times with PBSt
5. Wash the beads 2 times with 0.2M Sodium Borate pH9.0
6. With beads in Borate, prepare 20mM solution of dimethylpimilidate (DMP) in sodium borate (you will need a minimum of 10 fold excess volume of this solution relative to your starting bead bed volume, for example 50ul beads require a minimum of 500 µl of 20mM DMP solution).
7. Resuspend beads in at least a ten fold excess of Borate + 20mM DMP.  If I have less than 100µl of beads, I usually use 500-1000µl regardless.
8. Rotate at room temperature for 30 minutes.
9. Spin down beads and wash 2-3 times in 100mM Tris pH8. This step quenches the coupling reaction. 
10. Allow beads to rotate with tris for 2 hours at room temperature, or overnight in the cold room.
11. Wash beads 2-3 times in PBSt
12. Wash beads 3 times in 0.1M glycine pH2.5.  This step removes any unbound antibody. 
13. Wash beads back into PBSt 3 times. The glycine will effectively denature the antibodies bound to beads, so it is important to let them refold in PBSt for a minimum of several hours to overnight in the cold.  If not the IP can be very dirty.
14. Wash beads in buffer of choice prior to IP.  Spin down and remove excess buffer. 
15. Add solution you are IP-ing from to the beads, resuspend and incubate at either room temperature or in cold for a minimum of 1-2 hours.  The length of time for the IP depends on the quality of the antibody.  Also, things will get degraded faster (much faster) at room temperature.  I would recommend starting with 2-3 hours in the cold.
16. After the IP, wash the beads 3 times in buffer (PBSt works, although you can wash with higher salt to be more stringent). 
17. Elute the beads 3 times in 0.1M Glycine pH 2.5.  Transfer the supernatant after each elution into 1.0M Tris pH8.  Normally, I will elute with 3 times, each with 200-300µl of glycine, and put all three elutions into 300µl of 1M Tris (final volume of 1.2ml).
18. TCA precipitate samples by adding a 1:50 volume of 1% DOC and a 1:10 volume of TCA. Vortex quickly to mix. 


5x H1 Kinase Buffer - Amount per reaction: 2µl Final Concentration: 80mM b-Glycerophosphate, 12mM MgCl2, 15mM EGTA pH 7.4
19. Store TCA precipitation on ice for 30 minutes to overnight (30 minutes is almost always sufficient).  


ATP (10mM Stock) - Amount per reaction: 0.1 µl Final Concentration:100 µM
20. Spin in a cold benchtop centrifuge at high speed for 15 minutes.  


g32-P ATP - Amount per reaction: 0.1 µl
21. Remove supernatant, add 500µl of acetome to pellet to remove salt and crap. 


Histone H1 (10mg/ml Stock) - Amount per reaction: 0.125 µl  Final Concentration: 1.25 µg
22. Respin for 5-10 minutes on high speed.


H2O 7.675 µl
23. Remove all supernatant and resuspend in sample buffer.  If sample buffer doesn’t stay blue, that is bad.
Add LPC to 10µg/ml, DTT to 1mM and NP-40 Substitute to 0.1%.  I usually mix NP-40 and water first and vortex into solution. 
5x H1 Kinase Buffer is stored in aliquots at -20°C.  
   
Histone H1 is made up in water and stored at -20°C
Keep the mixture on ice until a few minutes before beginning the experiment.  At this point place at room temperature for about 5 minutes, allowing it to warm up. 
Extract samples are moved from the -80°C freezer into an ice bucket immediately before beginning the experiment.  Reactions are started by the addition of 9µl of the H1 Kinase mixture (above).  Pipette up and down to mix the kinase assay solution and the extract, and incubate at room temperature for 30 minutes.  The reactions are stopped by the addition of 20µl of 2x Laemmli Sample Buffer.  Samples are run on SDS-PAGE gels, coomassie stained, destained, dried, and placed on a phosphoimager. 
  1.  Chen, R.H. and A.Murray. 1997. Characterization of spindle assembly checkpoint in Xenopus egg extracts. Methods Enzymol. 283:572-584.

Latest revision as of 13:32, 15 November 2011

Protocol submitted by VGP from Stukenberg lab protocols [1]


link to protocol page [2]


IP Protocol

This protocol utilizes the coupling of Antibody to Protein A Coated beads using DMP. This allows for elution of bound proteins and reuse of beads, if desired.


Materials:

Bio-Rad Protein A Support

Control IgG (depending on antibody source; rabbit, mouse, etc..)

PBSt (PBS + 0.1% Tween20)

0.2 M Sodium Borate pH 9.0

0.1M Glycine, pH 2.5

Trichloroacetic Acid (TCA)

1% Deoxycholic acid (DOC)


Methods

1. Remove an appropriate amount of Protein A beads and wash 3 times with PBSt. You will need a minimum of 2 tubes of beads at this point, one for you’re your antibody of interest and one for control IgG.

2. Assuming you are using 10-100 µl of beads, add PBSt and antibody to a final volume of ~500µl.

3. Mix the antibody/bead solution in the cold room for 1 hour to overnight.

4. Wash the beads 3 times with PBSt

5. Wash the beads 2 times with 0.2M Sodium Borate pH9.0

6. With beads in Borate, prepare 20mM solution of dimethylpimilidate (DMP) in sodium borate (you will need a minimum of 10 fold excess volume of this solution relative to your starting bead bed volume, for example 50ul beads require a minimum of 500 µl of 20mM DMP solution).

7. Resuspend beads in at least a ten fold excess of Borate + 20mM DMP. If I have less than 100µl of beads, I usually use 500-1000µl regardless.

8. Rotate at room temperature for 30 minutes.

9. Spin down beads and wash 2-3 times in 100mM Tris pH8. This step quenches the coupling reaction.

10. Allow beads to rotate with tris for 2 hours at room temperature, or overnight in the cold room.

11. Wash beads 2-3 times in PBSt

12. Wash beads 3 times in 0.1M glycine pH2.5. This step removes any unbound antibody.

13. Wash beads back into PBSt 3 times. The glycine will effectively denature the antibodies bound to beads, so it is important to let them refold in PBSt for a minimum of several hours to overnight in the cold. If not the IP can be very dirty.

14. Wash beads in buffer of choice prior to IP. Spin down and remove excess buffer.

15. Add solution you are IP-ing from to the beads, resuspend and incubate at either room temperature or in cold for a minimum of 1-2 hours. The length of time for the IP depends on the quality of the antibody. Also, things will get degraded faster (much faster) at room temperature. I would recommend starting with 2-3 hours in the cold.

16. After the IP, wash the beads 3 times in buffer (PBSt works, although you can wash with higher salt to be more stringent).

17. Elute the beads 3 times in 0.1M Glycine pH 2.5. Transfer the supernatant after each elution into 1.0M Tris pH8. Normally, I will elute with 3 times, each with 200-300µl of glycine, and put all three elutions into 300µl of 1M Tris (final volume of 1.2ml).

18. TCA precipitate samples by adding a 1:50 volume of 1% DOC and a 1:10 volume of TCA. Vortex quickly to mix.

19. Store TCA precipitation on ice for 30 minutes to overnight (30 minutes is almost always sufficient).

20. Spin in a cold benchtop centrifuge at high speed for 15 minutes.

21. Remove supernatant, add 500µl of acetome to pellet to remove salt and crap.

22. Respin for 5-10 minutes on high speed.

23. Remove all supernatant and resuspend in sample buffer. If sample buffer doesn’t stay blue, that is bad.

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