Immunoprecipitation (Stukenberg lab) and Silver staining protein gels (Stukenberg lab): Difference between pages

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IP Protocol
Silver Staining Protocol (Not Compatible with Mass Spec)
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
Notes:
Be careful not to touch gel during any step of the procedure with either a gloved or ungloved hand, finger etc…
Use MilliQ-H2O for all steps
Perform all incubations in well washed glass dishes.


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


PBSt (PBS + 0.1% Tween20)
1. Remove gels from glass plates in which they were run:
 
0.2 M Sodium Borate pH 9.0
2. Incubate in 50% Methanol, 10% Glacial Acetic Acid, 40% H2O for 30 minutes (100 ml)
 
0.1M Glycine, pH 2.5
3. Incubate in 5% Methanol, 7% Acetic Acid (Glacial), 88% H2O for 30 minutes (100ml)
 
Trichloroacetic Acid (TCA)
4. Incubate in 10% Gluteraldehyde made up in H2O from 25% stock (25 ml) for 30 minutes.
 
1% Deoxycholic acid (DOC)
5. Wash 4 x 20-30 minutes in H2O using 200-300 ml per wash.
6. Makes 2 Solutions, A and B:    A) 3 ml of 30% Ammonia Hydroxide, 0.25 g NaOH in 190 ml H2O.  B) 1.2g Silver Nitrate (AgNO3) into 10 ml of H2O.
7. Add solution B to A slowly, mix gently, and add to gel, incubate for 30 minutes.
8. Make developer and stop solutions (see below).
9. Perform 5 x 1 minute washes with H2O, using 200 ml’s per wash.
   
   
10. Add developer solution (~100 ml) and wait and watch gel until you see bands come up.  Quickly remove developer solution and add stop solution.  (Developer Solution: 0.1g Citric Acid, 1ml 37% formaldehyde into 1L H2O. Stop Solution: 190 ml H2O and 10 ml Acetic Acid, Glacial)
   
   
Methods
11. After 10 minutes in Stop Solution wash thoroughly
 
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.

Revision as of 10:11, 14 November 2011

Protocol submitted by VGP from Stukenberg lab protocols [1]


link to protocol page [2]


Silver Staining Protocol (Not Compatible with Mass Spec)


Notes: Be careful not to touch gel during any step of the procedure with either a gloved or ungloved hand, finger etc… Use MilliQ-H2O for all steps Perform all incubations in well washed glass dishes.


1. Remove gels from glass plates in which they were run:

2. Incubate in 50% Methanol, 10% Glacial Acetic Acid, 40% H2O for 30 minutes (100 ml)

3. Incubate in 5% Methanol, 7% Acetic Acid (Glacial), 88% H2O for 30 minutes (100ml)

4. Incubate in 10% Gluteraldehyde made up in H2O from 25% stock (25 ml) for 30 minutes.

5. Wash 4 x 20-30 minutes in H2O using 200-300 ml per wash.

6. Makes 2 Solutions, A and B: A) 3 ml of 30% Ammonia Hydroxide, 0.25 g NaOH in 190 ml H2O. B) 1.2g Silver Nitrate (AgNO3) into 10 ml of H2O.

7. Add solution B to A slowly, mix gently, and add to gel, incubate for 30 minutes.

8. Make developer and stop solutions (see below).

9. Perform 5 x 1 minute washes with H2O, using 200 ml’s per wash.

10. Add developer solution (~100 ml) and wait and watch gel until you see bands come up. Quickly remove developer solution and add stop solution. (Developer Solution: 0.1g Citric Acid, 1ml 37% formaldehyde into 1L H2O. Stop Solution: 190 ml H2O and 10 ml Acetic Acid, Glacial)

11. After 10 minutes in Stop Solution wash thoroughly

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