Dynabeads Co-Immunoprecipitation Kit

Intended Use

The Dynabeads® and buffers provided in this kit will enable you to a) covalently immobilize antibodies of your choice onto the surface of Dynabeads, and b) use the antibody coated beads for co-immunoprecipitation. This kit is intended to be used for the co-immunoprecipitation of proteins, intact protein complexes, intact protein-nucleic acid complexes, etc. The antibody coated Dynabeads surface has ultra-low background binding, thus does not require blocking prior to use. The protein to bead interaction occurs directly on the bead surface; this enables the ultra rapid procedure that permits isolation of even labile composites, and allows isolation of complexes of all sizes (i.e. no upper size limit). This kit is designed for and tested with cultured yeast and mammalian cells and is compatible with tissue, insect, bacteria, and other lysates. For best results, please read through the manual carefully prior to start.

Principle

Antibodies (Ab) of your choice are covalently coupled to Dynabeads. Once antibodies have been coupled to the Dynabeads, the bead-bound antibody may then be used for co-immunoprecipitation experiments. Captured proteins and protein complexes are easily separated from lysate using magnetic separation properties of Dynabeads. Magnetic separation facilitates washing, buffer changes, and elution. Antibody-to-bead coupling works optimally with purified antibodies, although the coupling reaction also works well with antibodies in storage buffers that include protein additives (e.g. BSA) and/or sodium azide (NaN₃). This kit is not recommended for use with antibodies that have been stabilized in glycerol.

Materials Supplied

Reagents are analytical grade and are compatible with protease & phosphatase inhibitors. All kit components can be stored between 2 °C and room temperature.

Dynabeads® M-270 Epoxy (> 60 mg)

• C1 (20 ml)                  • LB (15 ml)
• C2 (8 ml)                    • HB (15 ml)
• 5 × IP (120 ml)            • SB (40 ml)
• 5 × LWB (24 ml)         • EB (4 ml)

Additional Materials Required

• Magnet: e.g. DynaMag™-2   (see www.lifetechnologies.com/magnets).
• Mixer allowing rotation or tilting of tubes.
• Antibodies of your choice.

Optional: Device for Cryolysis

Optional: Buffer Modifiers:

• Sodium Chloride (e.g. 1M NaCl)
• Dithiothreitol (e.g. 1M DTT)
• Magnesium Chloride (e.g. 1M MgCl₂)
• Potassium Acetate (e.g. 1M KOA_c)
• Tween®-20
• Triton™ X-100
• Protease inhibitors without EDTA

Optimal Antibody Use

Antibody coupling to Dynabeads is most efficient when using low quantities of antibody per mg beads. For applications such as immunoprecipitation and co-immunoprecipitation, the recommended amount of antibody for a coupling reaction is typically 5 to 7 μg Ab/mg Dynabeads. For some assays saturation of beads with antibodies may be necessary. In such cases the excess antibodies may provide an increased blocking effect on the bead surface, potentially reducing nonspecific binding of certain proteins. However, excess antibodies will also increase nonspecific antibody interaction with non-target proteins. Moreover, antibody consumption will be significantly increased, along with the potential for (non-covalently) adsorbed antibody to “leak” off the beads. Therefore the optimal amount of antibody coupled per mg bead will depend upon the need for functional antibody balanced against the increased chance for non-specific antibody interaction and non-covalently bound antibody to leak off during the assay.

Antibody Selection

The choice of antibody is the most important factor for successful target capture. Note that not all antibodies are suitable for all applications. While a particular antibody may recognize and bind to its target antigen in some applications, such as Western Blotting, there is no guarantee that the same antibody will function well in immunoprecipitation or co-immunoprecipitation. Please refer to the antibody manufacturer’s recommendations regarding your antibody

Antibody Additives

Sodium Azide (NaN₃)

Many commercially available antibodies contain NaN₃ as preservative. The presence of NaN₃ can lead to a small decrease (<10%) in antibody coupling efficiency. This will not be a problem for most applications. Furthermore, if desired this can be easily compensated by slightly increasing the quantity of antibody used in the coupling reaction. Alternatively the NaN₃ can be removed prior to coupling by standard gel filtration chromatography or dialysis.

Antibody Stabilizing Proteins  

Some commercially available antibodies contain protein additives such as BSA or Gelatin. The presence of protein additives will not reduce antibody coupling efficiency if the total quantity of protein (antibody + protein additive) in the coupling reaction does not exceed the capacity of the beads. Protein additives present during the coupling reaction will be coupled to the bead surface along with antibody. In the case of BSA or Gelatin, the coupled proteins may provide a beneficial blocking effect but may also result in the co-isolation of BSA or Gelatin interacting proteins.

Glycerol

Coupling of antibodies or other proteins stabilized in glycerol is not recommended. Although it is possible to couple such ligands, the antibody (or protein) function may be severely affected.

Function and Stability of Antibodies and coupled to Dynabeads

Different antibodies have different characteristics. Even different antibody clones raised in the same species against the same antigen can vary in pI, antigen binding affinity, and stability. Consequently the coupling efficiency will vary between different antibodies. Furthermore, some coupled antibodies will retain their function for months, even years when stored properly, while others will lose their function within weeks. This is entirely antibody dependent.

Antibody Quantity

The amount of Dynabeads used for antibody coupling at any one time will depend upon the subsequent number and scale of co-immunoprecipitation samples to be run. Antibody coupling reactions should be scaled as outlined in Table 1. For best coimmunoprecipitation results, coupling of 5 μg to 7 μg antibodies per mg Dynabeads is recommended for high affinity antibodies. Coupling of more antibodies per mg Dynabeads may be necessary if the antibody affinity is poor. Typically, for co-immunoprecipitation of protein complexes with analysis by silver staining or Western blotting, 1.5 mg of antibody-coupled beads are used. For the detection by Coomassie staining, 7.5 mg of antibody coupled beads are used.

Antibody Aggregates and Antibody Leakage

The presence of antibody aggregates in the antibody stock used for coupling can result in antibody leakage during co-immunoprecipitation. To help reduce this we recommend that antibody aggregates be removed from the antibody stock by centrifugation at 16,000 × g for 10 min at 4ºC.

             Day 1

1. Weigh out the appropriate amount of Dynabeads® M-270 Epoxy (according to Table 1).
   
   Moisture on unused beads will deactivate the reactive groups necessary for covalent antibody coupling. To avoid condensation on unused beads, make sure the beads are at room temperature prior to opening the bottle.
   
   Table 1:  Calculation of Antibody and C1 Volumes
                                                                            
   

   		Volume        in         µl
   Beads (mg)	Antibody	C1	C2	Total Reaction Volume
   5	V	250-V	250	500
   10	W	500-W	500	1000
   20	X	1000-X	1000	2000
   40	Y	2000-Y	2000	4000
   60	Z	3000-Z	3000	6000

   
   Rule of Thumb: The total reaction volume (C1 - Ab + C2 ) should be 100 µl  per mg beads. The C1 + Ab volume is equal to C2 volume.
   


2. Wash the beads: Add 1 ml of C1 to the beads and mix by vortexing or pipetting.
   


3. Place the tube on a magnet and allow the beads to collect at the tube wall. Remove the supernatant.
   


4. Add the appropriate volume of antibody + C1 (according to Table 1) to the washed beads and mix by vortexing or pipetting.
   
   Example: If you are coupling 5 mg Dynabeads and your required quantity of antibodies has a volume of 100 μl, you will need to add 150 μl of C1 (i.e. 250μl C1 – 100μl Ab = 150μl.)


5. Add the appropriate volume of C2 and mix by vortexing or pipetting.


6. Incubate on a roller at 37°C overnight (16-24 hours). Make sure the fluid in the tube is mixing well. Make sure the beads do not settle. Beads settling during the overnight incubation will result in inefficient antibody coupling.

      Day 2


7. Place the tube on a magnet. Allow the beads to collect at the tube wall. Remove the supernatant.

Table 2: Required Buffer Volumes


		Volume        in         µl
Beads (mg)	HB	LB	SB	SB (for storage)
5	800	800	800	500
10	800	800	800	1000
20	1600	1600	1600	2000
40	1600	1600	1600	4000
60	1600	1600	1600	6000




8. HB Wash: Add 0.8 or 1.6 ml (Table 2) of HB and mix by vortexing or pipetting. Place the tube on a magnet, allow the beads to collect at the tube wall, then remove the supernatant.
   


9. LB Wash: Add 0.8 or 1.6 ml (Table 2) of LB and mix by vortexing or pipetting. Place the tube on a magnet, allow the beads to collect at the tube wall. Remove the supernatant.
   
   
10. Short SB Wash: Add 0.8 or 1.6 ml (Table 2) of SB and mix by vortexing or pipetting. Place the tube on a magnet, allow the beads to collect at the tube wall, then remove the supernatant. Repeat the wash once more.
    


11. Long SB Wash: Add 0.8 or 1.6 ml (Table 2) of SB and mix by vortexing or pipetting. Incubate on a roller/rotator at RT for 15 minutes. Place the tube on a magnet, allow the beads to collect at the tube wall, then remove the supernatant.
    


12. Resuspend beads in the same volume of SB as was the total coupling reaction volume and store at 4°C until use. The final bead concentration is 10 mg antibody coupled beads/ml. Your beads are now covalently coupled with antibody and ready for Co-IP.

1. Co-immunoprecipitate, using two different variants of Extraction Buffer (A and B)
.



Extraction Buffer A
Components	Final Conc.	Volume
5 X IP	1 X
NaCI	100 mM
Protease Inhib.
H20





Extraction Buffer B
Components	Final Conc.	Volume
5 X IP	1 X
NaCI	100 mM
MgCI2	2 mM
DTT	1 mM
Protease Inhib.
H20



Note that initially, both buffers are modified with 100 mM NaCl. Extraction Buffer B is further modified with 2 mM MgCl ₂ and 1 mM DTT.

Buffer Modifier Calculations:

[ ] mM Final Conc. × [ ] ml Total Buffer Vol. = [ ] ml Stock Used.
                                [ ] mM Stock Conc.

Example: To make 10 ml Extraction Buffer with 100 mM NaCl final concentration using a 2.5 M stock NaCl, the formula should look like following: 100 mM NaCl × 10 ml total Buffer / 2500mM NaCl stock = 0.4 ml NaCl stock




Based on the results from Step 1, a choice between Extraction Buffer A or B can be made. The buffer that yields the most numerous and distinct bands should be chosen for further optimization (provided that your complex consists of several proteins). If there is no difference between the two buffers, then use both in Step 2 in parallel.


a)   If you see too many bands and/or smeared bands, the buffer stringency is too low. In Step 2, use Extraction Buffer A or B (or both) with several different NaCl concentrations ranging from a 5  mM increment lower to several 50 mM increments higher (i.e. buffers with 50 mM, 100 mM, 150 mM, and 200 mM NaCl should be tested).
b)   If you see just your target protein and no other or very faint bands, the buffer stringency is too high. In Step 2, use Extraction Buffer A or B (or both) with several different NaCl concentrations ranging from a 25 mM increment higher to several 25 mM increments lower (ie buffers with 125 mM, 100 mM, 75 mM, 50 mM and 25 mM NaCl should be tested).  Based on the results from the Step 1, the abundance of your target protein can also be determined and the cell sample adjusted for subsequent immunoprecipitations.


The co-immunoprecipitation results obtained using Extraction Buffers with varying NaCl concentrations should, when examined on gels, show a gradient of lanes varying from too few and/or faint bands to too many and/or smeared bands. The optimal Extraction Buffer can thus be identified. If not, then continue NaCl optimization until such a gradient is seen.  In most cases NaCl concentrations of 200 mM and less will be required. Only in very rare cases will the NaCl concentration exceed 200 mM.  For most protein complexes, optimization of detergents is not necessary. However, detergent optimization may be necessary for certain membrane protein complexes. In such case, increasing concentrations of Triton X-100 is recommended as a first choice

Note that the 5 × IP diluted to 1 × already contains 0.5 % Triton X-100.



After Steps 2 and 3, the following should have been determined:

a) Choice between Extraction Buffer A or B

b) Optimal NaCl concentration

For further optimization, the NaCl may be exchanged for KOAc. Since Extraction Buffer A or B already contains 110 mM KOAc, KOAc increments of 10 mM should be tested (e.g. 110 mM, 120 mM, 130 mM and 140 mM). Once again, the co-immunoprecipitation results with Extraction Buffers with different KOAc concentrations should show a gradient of lanes which varies from too few and/or faint bands to too many and/or smeared bands from which the optimal condition may be identified.

Preparation of Samples for Co-Immunoprecipitation

The most appropriate method for the preparation of samples for co-immunoprecipitation will depend upon the type of sample to be used and the types of complexes to be isolated. In many instances cell disruption using detergent lysis is adequate. However, for isolation of large and/or labile complexes, whole cell cryolysis is strongly  recommended. Liquid N2 will stabilize labile complexes during the cryolysis process. Cryolysis protocols are applicable for all cell types. For further information on the recommended cryolysis procedure, please see References 1 to 4.

Detergent Lysis Method -  Mammalian Cell Culture Samples

Cell samples prepared using detergent lysis should be used immediately for co-immunoprecipitation. Make sure the antibody coupled beads from the Antibody Coupling Protocol section and the co-immunoprecipitation buffers are prepared prior to start.

1. Prepare an Extraction Buffer of the desired stringency by mixing ingredients according to the tables below. If necessary, adjust the concentration of DTT and protease inhibitors (see Co-Immunoprecipitation Optimization Section for details).



Extraction Buffer A
Components	Final Conc.	Volume
5 X IP	1 X
NaCI
Protease Inhib.
H20





Extraction Buffer B
Components	Final Conc.	Volume
5 X IP	1 X
MgCI2	2 mM
DTT	1 mM
Protease Inhib.
H20




2. Wash harvested cells once in PBS by centrifuging at 200 to 500 × g for 5 min at 4°C.


3. Resuspend cells in 1:9 ratio of cell mass to Extraction Buffer with protease inhibitors. Incubate on ice for 15 min. See Section 4.2 for details for calculation.
   
   Example: 50 mg cells are lysed in 450 μl buffer.
   


4. Centrifuge at 2600 x g for 5 min. at 4° C to remove large cell debris and nuclei


5. Transfer the supernatant to a fresh tube. Use immediately for co-immunoprecipitation.

Cryolysis Method - All types of cell types

A large batch of cell sample that can be used for multiple pull-down experiments can be prepared at once using this method. Approximately 6 L yeast or bacteria culture grown to OD600 ~ 0.8 is needed to produce ~ 20 g cells. Approximately 8 L of the mammalian cell line HEK 293 grown to late log phase is needed to produce ~ 12 g cells. Make sure all buffers and cells are kept on ice during the procedure. For the cryogenic freezing of the harvested cell sample, ~ 1 to 2 L liquid N₂ is needed. For tissue samples (e.g. liver) or whole organisms (e.g. Drosophila), weigh the sample directly and freeze in liquid N₂.

Use appropriate protective gear when working with liquid N₂. Contact your local environmental health work place safety officer for more information.

Preparation of Cell Samples for Cryolysis

1. Centrifuge 6 L yeast or bacteria culture or 8 L (or equivalent amount) mammalian or insect cell culture. Pellet the yeast or bacteria culture at 4000 × g and mammalian or insect cell culture at 500 × g for 10 min at 4°C. Discard the culture media. For mammalian and insect cells, skip Step 2; go directly to Step 3.


2. Wash the cells in 50 ml H₂O. Centrifuge yeast and bacteria cells at 2600 × g for 5 min at 4°C, then discard the supernatant.