c-Myc标签抗体偶联agarose beads（affinity gel）

 

Specificity： Anti- c-Myc Agarose beads(Affinity gel) is a mouse monoclonal antibody that is covalently attached to agarose beads by hydrazide linkage. The antibody binds c-Myc at the N-terminal, C-terminal and internal locations of fusion proteins.
Tested applications:  Immunoprecipitation (IP), 
40 ml of gel suspension (~20μl of packed beads) for ~1000μg of crude total protein solution.
Optimal dilutions/concentrations should be determined by the end user. 
Reactivity:  The antibody detects and captures c-Myc -Tagged fusion proteins overexpressed in mammalian cells or E.coli. 
Immunogen:  This monoclonal antibody is produced by immunizing mouses with a synthetic peptide (KLH-coupled) containing EQKLISEEDL.
Form:  The productis supplied as a 50% suspension in 50% glycerol with 10 mM sodium phosphate and 150 mM sodium chloride, pH 7.4, containing 0.02% (w/v) sodium azide
Binding capacity:  >10 nmoles of c-Myc-tagged fusion protein per 1 ml of settled gel.
Isotype:  Mouse IgG1
Recommended IP Lysis buffer for mammalian cells: 50 mM Tris HCl, pH 7.4, with 150 mM NaCl, 1 mM EDTA, and 1% TRITON X-100.
Storage instruction: 
The gel is supplied in 50% glycerol, so the gel can be stored at –20 °C for maximum stability. The unopened product is stable for one year when stored as indicated.
Attention: Agarose Beads should be resuspended well before used in IP

Immunoprecipitation(IP) protocol
 

1. Sample Lysate Preparation

It is recommended that the CelLytic B Lysis Reagents (sigma, Catalog Numbers B7435, B7310, or C8740) or CelLytic B Plus Kit (sigma, Catalog Numbers CB0050 or CB0500) be used for bacterial lysis.
IP Lysis buffer (50 mM Tris HCl, pH 7.4, with 150 mM NaCl, 1 mM EDTA, and 1% TRITON X-100), or CelLytic M (sigma, Catalog Number C2978) can be used for mammalian cells.

A. Recommended procedure for E. coli using CelLytic Lysis Reagents
1. Grow the cells (~1 liter or less) under conditions that induce production of c-Myc fusion proteins.
2. Harvest the cells by centrifugation at 5,000×g for 30 minutes at 2-8 °C.
3. Decant the medium from the cell paste.
4. Freeze the cell paste using a dry ice/ethanol bath or at –20 °C in a freezer. Cell lysis is enhanced during the slow freezing.
5. Lyse the frozen cells with 10 ml of CelLytic B (sigma, Catalog Number B7435) per g of frozen cell paste or 5 ml of CelLytic B, 2×concentrate (sigma, Catalog Number B7310) per g of frozen cell paste.
6. Resuspend the cells in the CelLytic B reagent with a pipette. Mix vigorously on a stir plate for 15 minutes to fully extract the protein.
7. Remove the cell debris by centrifuging for 15 minutes at 21,000×g.
8. After centrifugation, decant the supernatant into a fresh container and dispose of the cell pellet. The solution should be clear with no insoluble particles.

B. Recommended procedure for mammalian cells
For a 70–90% confluent 100 mm dish (10⁶–10⁷ cells), use 1 ml of IP lysis buffer (50 mM Tris HCl, pH 7.4, with 150 mM NaCl, 1 mM EDTA, and 1% TRITON X-100). If the expression level of the c-Myc fusion protein is relatively low, lyse the cells with a reduced volume of IP lysis buffer. It is highly recommended to add a protease inhibitor cocktail (sigma, Catalog Number P8340) to the IP lysis buffer (10 ml per 1 ml of lysis buffer), especially if the lysate is to be stored for further use.
1. Wash adherent or suspension cells as appropriate:
Adherent Cells - Remove the growth medium from the cells to be analyzed. Rinse the cells twice with PBS buffer, being careful not to dislodge any of the cells. Discard the PBS. Add lysis buffer (10⁶–10⁷cells/ml).
Cells in Suspension - Collect the cells into an appropriate conical centrifuge tube. Centrifuge for 5 minutes at 420×g. Decant the supernatant and discard. Wash the cells twice by resuspending the cell pellet with PBS and centrifuge for 5 minutes at 420×g. Decant the supernatant and discard. Resuspend the cell pellet in lysis buffer (10⁶–10⁷  cells/ml).
2. Incubate the cells for 15–30 minutes on a shaker.
3. For adherent cells only, scrape and collect the cells. For cells in suspension, proceed to step 4.
4. Centrifuge the cell lysate for 10 minutes at 12,000×g.
5. Transfer the supernatant to a chilled test tube. For immediate use, keep on ice. If the
supernatant is not to be used immediately, store it at –70 °C.

2. Pre washing the gel beads to remove glycerol from product buffer.
The anti- c-Myc affinity gel is stored in 50% glycerol with buffer. The glycerol must be removed just prior to use and the gel equilibrated with buffer.
1. Thoroughly resuspend the anti- c-Myc Agarose gel by inverting the product tube or by pipette tip. Note: this step is very important. Make sure to resuspend the gel very well.
2. Immediately transfer 40ml of the gel suspension to a fresh tube. Then add 0.5ml TBS (50 mM Tris HCl, with 150 mM NaCl, pH 7.4), centrifuge the gel suspension at 5,000–8,000×g for 30 seconds, remove the supernatant carefully, make sure that most of the wash buffer is removed and no gel beads is discarded. This step should be repeated for 3-4 times.
In case of numerous immunoprecipitation samples, wash the gel beads needed for all samples together. After washing, divide the gel according to the number of samples tested. Each wash should be performed with TBS at a volume equal to 20 times the total packed beads volume.
Note: For the gel transfer, use a clean, plastic pipette tip with the end enlarged to allow the gel to be transferred easyly.

3. c-Myc Fusion Protein Immunoprecipitation
1. Add 200–1,000 ml of E.coli extracts or cell lysates to the washed gel. If necessary, bring the final volume to 1 ml by adding IP lysis buffer (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% TRITON X-100).
Note: The volume of E.coli extracts or cell lysates to be used depends on the expression level of c-Myc fusion protein in the transfected cells or bacterial. The amount of c-Myc fusion protein to be precipitated depends on the detection method. 200 ng of protein is sufficient for an activity assay or for an immunoblot analysis. For SDS-PAGE analysis with Coomassie blue or silver staining, use 1 mg of c-Myc fusion protein. 
For the negative control, add only 1 ml of IP lysis buffer with no protein.
2. Agitate or shake all samples and controls gently (a roller shaker is recommended) for 2 hours. In order to increase the binding efficiency, the binding step may be extended overnight.

4. Washing immune complexes to remove nonspecific binding proteins.
1. Centrifuge the resin for 30 seconds at 5,000–8,000×g. Remove the supernatants with a narrow-end pipette tip.
2. Wash the gel three times with 0.5 ml of TBS (50 mM Tris HCl, with 150 mM NaCl, pH 7.4). Make sure all the supernatant is removed by using a Hamilton syringe or equivalent device.

5. Elution and obtaining of the c-Myc -fusion protein
Two elution and obtaining methods are recommended according to protein characteristics or further usage:
Option A: Elution under acidic conditions with 0.1 M glycine HCl, pH 3.5. This is a fast and efficient elution method. Equilibration of the eluted proteins with neutralizing buffer (0.5 M Tris HCl, with 1.5 M NaCl, pH 7.4) may help preserve its activity.

Option B: Boil with sample loading buffer, Centrifuge and get the supernatants for gel electrophoresis and western blotting.

Option A: Elution with 0.1 M glycine HCl, pH 3.5 
The procedure should be performed at room temperature. 
Note: Do not leave the gel beads in this buffer more than 20 minutes.
1. Add 100 ml of 0.1 M glycine HCl buffer (pH 3.5) to each sample and control gel.
2. Incubate the samples and controls with gentle shaking for 5 minutes at room temperature.
3. Centrifuge the gel for 60 seconds at 8,000×g. 
4. Transfer the supernatants to fresh test tubes using a Hamilton syringe or equivalent device. Be careful not to transfer any gel beads.
5. In order to equilibrate the eluant, immediately add 10 ml of neutralizing buffer (0.5 M Tris HCl, with 1.5 M NaCl, pH 7.4) to each sample.
6. For immediate use, for example, western blotting, store the eluted proteins at 2–8 °C. Store at –20 °C for long term storage.
For downstream western blotting analysis, add appropriate SDS-PAGE Sample loading Buffer to the eluted proteins and boil, then the samples and controls are ready for loading on SDS-PAGE and immunoblotting using anti-c-Myc or specific antibodies against the fusion protein.

Option B: Boil with sample loading buffer, Centrifuge and get the supernatants for gel electrophoresis and western blotting.
The procedure should be preformed at room temperature. Sample loading buffer should be at room temperature before use. In order to minimize the denaturation and elution of the antibody, no reducing agent (2-mercaptoethanol or DTT) should be included in the Sample loading buffer. The addition of reducing agents will result in the dissociation of the heavy and light chains of the immobilized anti c-Myc antibody (25 and 50 kDa bands). If reducing conditions are absolutely necessary, a reducing agent may be added. The final concentration of 2-mercaptoethanol or DTT in the 1×sample loading buffer (62.5 mM Tris HCl, pH 6.8, with 2% SDS, 10% (v/v) glycerol, and 0.002% bromphenol blue) should be 5% or 50 mM, respectively.
Note: SDS in the sample buffer will denature the anti c-Myc antibody, and the anti c-Myc affinity gel cannot be reused after treatment with the SDS-PAGE Sample loading buffer.
1. Add 20 ml of 2×sample loading buffer (125 mM Tris HCl, pH 6.8, with 4% SDS, 20% (v/v) glycerol, and 0.004% bromphenol blue) to each sample and control.
2. Boil the samples and controls for 3 minutes.
3. Centrifuge the samples and controls at 8,000×g for 60 seconds to pellet any undissolved agarose beads. Transfer the supernatants to fresh test tubes with a Hamilton syringe or a narrow-end Pasteur pipette. The samples and controls are ready for loading on SDS-PAGE and immunoblotting using anti- c-Myc or specific antibodies against the fusion protein.

6. SDS-PAGE for western blotting.

Precautions and Disclaimer
This product is for R&D use only, not for drug, household, or other uses. 

Reagent Incompatibility Table
 

Reagent	Effect	Notes
Chaotropic agents (e.g., urea, guanidine HCl)	Denatures the immobilized anti c-Myc antibody	Do not use any reagent that contains these types of components since it will denature the anti c-Myc antibody on the gel and destroy its ability to bind the c-Myc fusion proteins.Low concentrations of urea (1 M or less) can be used.
Reducing agents (such as DTT, DTE, 2-mercaptoethanol)	Reduces the disulfide bridges holding the anti c-Myc antibody chains together	Do not use any reagent that contains these types of components since it will reduce the disulfide linkages in the anti c-Myc antibody on the gel and destroy its ability to bind the c-Myc fusion proteins.
Sodium dodecyl sulfate (SDS)	Denatures the immobilized anti c-Myc antibody	Do not use any reagent that contains this detergent in the lysis and washing buffers since it will denature the anti c-Myc antibody on the gel and destroy its ability to bind the c-Myc fusion proteins. It is included in the sample loading buffer for removal of protein for immunoprecipitation, but the gel cannot be reused.
Deoxycholate	Interferes with anti c-Myc antibody  binding to c-Myc proteins	Do not use any reagent that contains this detergent since it will inhibit the anti c-Myc antibody from binding to c-Myc fusion proteins.

Reagent Compatibility Table
 

Reagent	Effect	Notes
TWEEN 20, 5% or less	Reduces nonspecific protein binding to the gel	May be used up to recommended concentration of 5%, but do not exceed.
TRITON X-100 5% or less	Reduces nonspecific protein binding to the gel	May be used up to recommended concentration of 5%, but do not exceed.
IGEPAL CA-630, 0.1% or less	Reduces nonspecific protein binding to the gel	May be used up to recommended concentration of 0.1%, but do not exceed.
CHAPS, 0.1% or less	Reduces nonspecific protein binding to the gel	May be used up to recommended concentration of 0.1%, but do not exceed.
Digitonin, 0.2% or less	Reduces nonspecific protein binding to the gel	May be used up to recommended concentration of 0.2%, but do not exceed.
Sodium chloride, 1.0 M or less	Reduces nonspecific protein binding to the gel by reducing ionic interactions	May be used up to recommended concentration of 1.0 M, but do not exceed.
0.1 M glycine HCl, pH 3.5	Elutes c-Myc protein from the gel	Do not leave the beads in glycine HCl for longer than 20 minutes. Longer incubation times will begin to denature the anti c-Myc antibody

Troubleshooting Guide
 

Problem	Possible Cause	Solution
No signal is observed.	c-Myc fusion protein is not present in the sample.	1. Make sure the protein of interest contains the c-Myc -tag by immunoblot or dot blot analyses.
		2. Prepare fresh lysates. Avoid using frozen lysates.
		3. Use appropriate protease inhibitors in the lysate or increase their concentrations to prevent degradation of the c-Myc fusion protein.
	Washes are too stringent.	1. Reduce the number of washes.
		2. Avoid adding high concentrations of NaCl to the mixture.
		3. Use solutions that contain less or no detergent.
	Incubation times are inadequate.	Increase the incubation times with the affinity gel (from several hours to overnight).
	Interfering substance is present in sample.	1. Lysates containing high concentrations of dithiothreitol (DTT), 2-mercaptoethanol, or other reducing agents may destroy antibody function, and must be avoided.
		2. Excessive detergent concentrations may interfere with the antibody-antigen interaction. Detergent levels in buffers may be reduced by dilution.
	Detection system is inadequate.	If Western blotting detection is used: