Chaps Structure


A nondenaturing zwitterionic detergent for solubilizing membrane proteins and breaking protein-protein interactions. Combines the useful properties of both the sulfobetaine-type and the bile salt detergents. Commonly used for protein solubilization in isoelectric focusing and two-dimensional electrophoresis especially for non-denaturing (without urea) isoelectric focusing. CHAPS have been shown to give excellent resolution of some subcellular preparations and plant proteins. Concentrations between 1-4% (v/v) are typically used in an isoelectric focusing gel. A commonly used isoelectric focusing sample solution consists of 8 M urea, 4% CHAPS, 50-100 mM dithiothreitol (DTT) and 40 mM Tris. Its small micellar molecular weight (6150) and high CMC (6-10 mM) allow it to be removed from samples by dialysis. It can be used as a non-toxic stabilizing agent for growth factors.

Molecular Formula: C32H58N2O7S
Molecular Weight: 614.9
Density: 1.01g/mL at 20 °C
CAS #: 75621-03-3
Synonyms: 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane sulfonate; N, N-Dimethyl-N-(3-sulfopropyl)-3-[[(3a, 5b, 7a, 12a)-3, 7, 12-trihydroxy-24-oxocholan-24-yl] amino]-1-propanaminium inner salt

1. What is the physical appearance of CHAPS?
White crystalline powder

2. What is the critical Micelle Concentration (CMC) of CHAPS?
6-10 mM (Detergents with high CMC values are generally easy to remove by dilution; detergents with low CMC values are advantageous for separations on the basis of molecular weight. As a general rule, detergents should be used at their CMC and at a detergent-to-protein weight ratio of approximately ten.

3.In 0-0.1M Na+, what is the Aggregation Number?

4. What is the transition temperature?
cloud point>100 °C

5. What is the TLC (Silica gel: methanol/NH4OH=95/5) of CHAPS?
One spot.

6. Is CHAPS soluble in water?
Yes. It becomes clear, colorless to slightly yellow solution in water. Solubility is 50mg/ml at 20 °C.

7.When preparing solutions of CHAPS, how about shaking it to mix?
No. It is preferable to avoid stirring and/or shaking.

8. How can I make 10% solution of CHAPS?
For a 10% solution, i.e., 1 g CHAPS solid into beaker followed by 9 g of water. Cover beaker with watch glass and allow it to sit at room temperature for 30-60 minutes. Solutions of up to 1 M (60%) can be made in this way.

9. What is the stability of CHAPS?
It is moisture sensitive and hydroscopic.

10. Can be CHAPS removed from samples by dialysis?
Yes. CHAPS’s small micellar molecular weight and high critical micelle concentration (6-10mM) allow it to be removed from samples by dialysis.

11. What is the difference between CHAP and CHAPSO detergent?
A related detergent, called CHAPSO, has the same basic chemical structure with an additional hydroxyl functional group. Both detergents have low light absorbance in the ultraviolet region of the electromagnetic spectrum, which is useful for laboratory workers monitoring ongoing chemical reactions or protein-protein binding with UV/Vis spectroscopy.

Chaps 2


Chemical Table

12. What concentration of CHAPS is used for preparing IEF gel?
Concentrations between 2-4 %( wandv) are typically used in an IEF gel. CHAPS is commonly used for non-denaturing (without urea) IEF and has been shown to give excellent resolution of some subcellular preparations and plant proteins.

13. What is the difference between Triton X100 and CHAPS detergent?
There are multiple differences. Triton X-100 is non-ionic. It has both hydrophilic and hydrophobic regions, but no net charges. CHAPS is zwiterionic. It has hydrophobic regions but also a head group with a negative charge (in normal saline). In addition, Triton X100 forms large (greater than 90,000 MW) aggregates when TGriton X100 concentration rises above 0.25 mM. CHAP on the other hand forms smaller aggregates (6,000 MW) when CHAPS concentration rises above 10 mM.

14. Commonly used detergents?
Detergents are polar lipids that are soluble in water. The presence of both a hydrophobic and hydrophilic portion makes these compounds very useful for lyses of lipid membranes, solubilization of antigens, and washing of immune complexes
Types of Detergents A large number of detergents are available for scientific use. These are usually categorized according to the type of hydrophilic group they contain - anionic, cationic, amphoteric, or nonionic (see Table 1). In general, nonionic and amphoteric detergents are less denaturing for proteins than ionic detergents. Sodium cholate and deoxycholate are the least denaturing of the ionic detergents of commonly used detergents.

Two properties of detergents are important in their consideration for biological studies: the critical micelle concentration (CMC) and the micelle molecular weight. The CMC is the concentration at which monomers of detergent molecules combine to form micelles. Each detergent micelle has a characteristic molecular weight. Detergents with a high micelle molecular weight, such as nonionic detergents, are difficult to remove from samples by dialysis. The CMC and the micelle molecular weight vary depending on the buffer, salt concentration, pH and temperature. In general, adding salt will lower the CMC and raise the micelle size.

Table 1 - Physical Properties of Commonly Used Detergents

Table 1

 Table 2 - Chemical Properties of Commonly Used Detergents

Table 2

Choice of Detergents

Ionic detergents are very good solubilizing agents, but they tend to denature proteins by destroying native threedimensional structures. This denaturing ability is useful for SDS-PAGE but is detrimental where native structure is important for functional activity. It should be noted that antibodies usually retain their binding.

Activity at 0.1% SDS or less. Nonionic and mildly ionic detergents are less denaturing and can often be used to solubilize membrane proteins while retaining protein-protein interactions. The following detergents have detrimental properties for some procedures:

1. Phenol containing detergents (i.e., Triton X-100 and NP40) have a high absorbance at 280nm and thus interfere with protein monitoring at that OD. Lubrol may be substituted. Phenol-containing detergents also induce precipitation in the Folin (Lowry) protein assay. They can also be iodinated...and thus should not be used if iodinating proteins.

2. High micellar molecular weight interferes with gel filtration and not easily removed by dialysis.

3. Sodium cholate and sodium deoxycholate are insoluble below pH 7.5. Above an ionic strength of 0.1%SDS they will often crystallize.

4. Ionic detergents interfere with nondenaturing electrophoresis and isoelectric focusing.

Chaps Cell Extract Buffer
Chaps Cell Extract Buffer can be used to lyse cells under nondenaturing conditions and is recommended for the preparation of cytoplasmic cell lists to be used with our caspase signaling pathway antibodies.

Components: 10X CHAPS Cell Extract Buffer (5ml)

1X concentration:50 mM Pipes/HCl (pH 6.5), 2 mM EDTA, 0.1% Chaps, 20 μg/ml Leupeptin, 10 μg/ml Pepstatin A,10 μg/ml

  • Aprotinin. Add DTT to 5mM. 1 M (200X) DTT (0.25 mL)
  • 1X concentration: 5 mM DTT
  • Preparation of 1X Cell Extract Buffer:
  1. Buffer should be prepared immediately before use.
  2. Immediately before dilution, invert the 10X Chaps Cell Extract Buffer several times to suspend all buffer components.
  3. Add 1/10 volume of 10X Chaps Cell Extract Buffer to 9/10 volume of Milli-Q water or equivalently purified water.
  4. Add DTT to final concentration of 5 mM (1:200 dilution) and PMSF* to final concentration of 1 mM.

Sample Preparation Using Chaps Cell
Extract Buffer:
(a) Treat cells by adding fresh media containing regulator for desired time.
(b) Aspirate media from cultures; wash cells three times with PBS; aspirate. Scrape cells into PBS and spin down to pellet.
(c) Lyse cells by adding ice cold 1X Chaps Cell Extract Buffer (1 volume of cell pellet). Resuspend cells in the buffer, freeze at -80°C and thaw twice, centrifuge the lysate cold at 14,000 rpm. Keep the supernatant and discard the pelleted cell debris.
(d) Add SDS Sample Buffer and heat sample to 95–100°C for 5 minutes; cool on ice.
(e) Microcentrifuge for 5 minutes.
(f) Load 5 μL onto SDS-PAGE gel (10 cm x 10 cm).
Storage: Store Chaps Cell Extract Buffer and 200X dithiothreitol (DTT) at -20°C.

Cell Lysis and Immunoprecipitation Protocol with CHAPS Buffer

Part No. CIB-1,1X Solution, pH 8.0; 60ml or 120ml Background The CHAPS Immunoprecipitation Buffer (CIB-1) is formulated to maintain intermolecular interactions following transmembrane and cytosolic protein extraction. The CHAPS buffer is ideal for “pull down-co-immunoprecipitation” (i.e.Co-IP) analysis, since it maintains protein conformation as well as protein complex assembly.

Following cell lysis and cellular protein solubilization into CHAPS buffer, assembled proteins can be isolated together using immunoprecipitation technique. An immunoprecipitating antibody is added to bind to one of the assembled protein targets. A solid support, such a Protein-G Sepharose, is then added to non-covalently link and bind to the antibody-protein complex and make it insoluble. Following low speed centrifugation, sedimentation and wash, the solid support-antibody-protein complex is isolated together. Proteins assembled with the protein targeted by the immunoprecipitating antibody become co-immunopreciptated. By developing Western blots with a different antibody targeting a co-assembled protein, the researcher can characterize and quantify the interaction among two or more protein species. Functional biochemical activity assays can also be employed to characterize co-iimmunoprecipitated and assembled biomolecules.

Required Equipment and Reagents:

1. Microcentrifuge at 4°C
2. Rocker Table
3. Cold room or refrigerated case.
4. Antibody suitable for immunoprecipitation as specified by the vendor. Generally, the immunoprecipitating antibody must recognize the NATIVE conformation.
5. Western blots applications: Antibody suitable for Western blot detection as specified by the vendor. This antibody must recognize the DENATURED state of the targeted protein.

Suggested Controls and Samples for Comparisons:

1. Perform immunopreciptation protocol with an irrelevant antibody to control for appearance of spurious protein bands.
2. Resolve in Western blots the original unfractionated cell lysate, the unbound fraction (e.g. the fraction containing proteins not bound to the protein-A or G solid support) and the immunoprecipitated materials to confirm fractionation and enrichment.
3. Develop a Western blot that detects the protein directly targeted by the immunoprecipitating antibody. This procedure confirms that immunoprecipitation occurred.




A. Cell Culture :
It is recommended that cells are cultured to 80-90% confluency prior to performing cell lysis and immunoprecipitation. Cells should be washed free of serum proteins using PBP prior to performing immunoprecipitation to prevent appearance of non-specific serum protein bands in downstream Western blots.

B. Cell Lysis:
We recommend using 300μl of CHAPS Buffer solution for one to three 10cm cell culture dishes of lysed cells. Scale accordingly for other numbers or sizes of cell culture dishes according to the surface area of the dish. Prior to lysis, make the CHAPS Buffer ice cold and add protease and phosphatase inhibitors.

1. Lyze cells and generate a supernatant fraction rapidly as follows: Apply the ice cold CHAPS Buffer solution to cells for 10 minutes: Use 300μl of CHAPS Buffer for one to three 10 cm plates of cells. Scale accordingly for other numbers or sizes of plates based on the surface area of the cell culture dish.

A simple method to dislodge and lyze adherent cells is to place the cell culture plates on a bed of ice. Dispense ice cold CHAPS Buffer with protease/phosphatase inhibitors over the cell layer, rotate the plate by hand to cover cells with a film of CHAPS Buffer, then immediately dislodge the cells with a cell scraper. Now, use a transfer pipette to siphon the cells into a 1.5ml microcentrifuge tube. The cell suspension can also be transferred sequentially over multiple plates to collect a concentrated cell suspension.
Note: Cells can also be collected by alternate methods into a 1.5ml tube, followed by lysis with the CHAPS Buffer as described below.

Place the 1.5ml micocentrifuge tube of cell suspension on ice for 10min; strongly tap the tube to facilitate cell membrane dissolution several times during this 10 min period. You can also use a rocker table to rotate the cell suspension to further facilitate cell membrane dissolution. Do not vortex the cell lysate if immunoprecipitation is planned.

2. Centrifuge the cell lysate in a cooled microcentrifuge at full speed for 15 min to partition supernatant and pellet. Collect the supernatant fraction, which contains extracted membrane and cytosolic proteins, and dispense this supernatant into another 1.5ml microcentrifuge tube that is placed in ice. This supernatant corresponds to the unfractionated supernatant fraction. Set aside a small aliquot for comparisons to the immunoprecipitated materials. The unfractionated supernatant can be stored at -20°C, or -80°C for longer term storage.

 C. Immunoprecipitation:

1. Add immunoprecipitating antibody to the unfractionated supernatant fraction, using the antibody titer recommended by the manufacturer. Place the tube with the immunoprecipitation reaction on a rocker table under refrigeration (such as a cold room, or refrigerated case) for 15-30 min. You may opt to initially try the shorter 15 min period for immunoprecipitation, a time period which is more apt to maintain low affinity interactions.

Directly dispense 60μl of immunoprecipitation beads (e.g.Sepharose-G beads, or protein-G beads) into every 300μl to 2ml aliquot of unfractionated supernatant with immunoprecipitating antibody. Place the tube on the rocker table for another 30 min to 1 hr under refrigeration to generate an insoluble solid support-antibody-protein complex.

Use a refrigerated microcentrifuge at 3000rpm for 5 minutes to sediment the immunoprecipitation beads. The resulting supernatant in this step is the unbound fraction. Collect the unbound fraction without disturbing the immunoprecipitating bead pellet. Store the unbound fraction supernatant at -20°C or -80°C.
Add an additional aliquot of fresh 300μl ice cold CHAPS Buffer with protease and phosphatase inhibitors to the immunoprecipitation beads. Gently rotate the tube 180° by hand three times and centrifuge again at 3000 rpm for 5 minutes. Remove and discard the wash supernatant. Repeat with two more washes of the immunoprecipitating beads in the same manner. After the last wash, use a microcentrifuge to sediment the immunoprecipitation beads at 14000 rpm for 15 min. Remove as much of the wash solution as possible using a pointed plastic Pasteur pipette. The immunoprecipitated fraction, which is bound to the beads, becomes sedimented at the bottom of the tube.

D. Elution of the Immunoprecipitated Fraction From the Beads and Western Blot:

1. There are several methods to elute the immuoprecipitated proteins from the immunoprecipitating beads to release a soluble immunoprecipitated fraction. The simplest method applicable for subsequent Western blotting is applying Laemmli Sample Buffer (LSB) directly to the immunoprecipitation beads: Add 100μl LSB for each 60μl of sedimented immunoprecipitation beads. Vortex the LSB-immunoprecititate solution at full speed for 30 sec, and then heat at 60°C for 10 min. Now sediment the beads using low speed centrifugation(3000 rpm) for 5 min. The immunoprecipitated proteins (along with the immunoprecipitating antibody) will be released into the supernatant; Collect this supernatant, which can be resolved in subsequent Western blots. Alternatively, if your protein of interest aggregates easily when heated in LSB, heat instead at 37°C for 30min. and follow the same aforementioned steps. In gels and Western blots, resolve in consecutive gel lanes, the unfractionated fraction, unbound fraction and immunoprecipitated fraction. With successful immunoprecipitation, you should observe enrichment of your protein of interest, along with any coassembled proteins.


1. Immunoprecipitation did not occur. Resolution: 1) You may have to empirically identify an antibody for immunoprecipitation that recognizes the native conformation of the epitope. 2) The protein complex is not maintained outside of a live-intact cell. Cross-linking procedures using membrane permeable cross-linking reagents and live cells may be required to capture interaction and assembly between two proteins.

2. The IgG heavy chain of the immunoprecipitating antibody (that was dispensed in the immunoprecipitation solution) migrates in gels at the same position as a protein of interest, and therefore masks its appearance in a Western blot. Resolution: For Western blot development, use an antibody derived from a different animal host from the immunoprecipitating antibody. In this case, the appropriate secondary-HRP conjugated antibody will not bind appreciably to the immunoprecipitating antibody, preventing the appearance of an overlaying Western blot band.