FT- GS2260
c-Myc
Mouse Monoclonal Antibody
Description
Catalog No:
GS2260
Clone Name:
9E11
Isotype :
Mouse IgG1
Antigen:
This antibody was raised against a synthetic peptide corresponding to amino acid residue(s)
408-420 of human c-Myc.
Reactivity:
Human
Buffer :
PBS containing 0.2% gelatin, 0.01% thimerosal and 0.1% sodium azide. Thimerosal and sodium
azide are highly toxic.
Storage:
Antibodies in solution can be stored at -80°C for 2 years. Avoid repeated freeze/thaw cycles and keep on
ice when not in storage. This product is guaranteed for 6 months from date of arrival.
Application
This antibody is suitable for Western blot. A 1:500 to 1:1000 dilution is
recommended. For optimal results, primary antibody incubations
should be performed at 37°C (detailed protocol included on page 2).
Individual optimization may be required.
Specificity
A band of approximately 61 kDa corresponding to c-Myc should be
detected in Western blot analysis.
Positive Control
K-562 nuclear extract can be used as a positive control for this antibody.
Detection of c-Myc by Western blot analysis. A
61 kDa form of c-Myc is detected in nuclear
extracts derived from K-562 cells using c-Myc
mouse monoclonal antibody. Lanes 1-3 represent
serial dilutions of the monoclonal antibody (1/500,
1/1000 and 1/2500).
For in vitro reasearch use only.
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P.1
FT- GS2630
Western Blotting protocol
Note: The addition of 0.1% Tween 20 to all Blotto solutions may increase the antibody specificity and decrease the
background.
In order to prevent bacterial growth, we also recommend that 0.01% thimerosal be added to all solutions with PBS.
When performing Western blotting experiments individual optimization of antigen quantities may be required in
order to detect the protein of interest. For example, the window of expression in different sources may result in
varying levels of intracellular factors and immunoreactivity is stronger when using recombinant proteins instead of
nuclear extracts.
Electrophoresis
1. Nuclear extracts are prepared according to Dignam's procedure1 and freeze aliquoted on liquid nitrogen. Prior to
electrophoresis, samples are thawed on ice and diluted in 4X sample buffer (250 mM Tris-HCl, pH 6.8, 8% SDS,
40% glycerol, 20% 2-mercaptoethanol, 2% bromophenol blue). Samples are then boiled 3-5 min., spun down, loaded
onto a 4-20% SDS-PAGE gel and run until migration front reaches the bottom of the gel.
We suggest that 20 µg of the recommended positive control be loaded into the appropriate well.
Transfer
2. Transfer protein to polyvinylidene difluoride (PVDF) membranes at 400 mA for 90 minutes using the buffer
described by Towbin et al2 (25 mM Tris, 192 mM glycine, 15% methanol).
Blocking
3. Block membranes by incubating overnight at 4°C with shaking in 5% Blotto (5% dry, non-fat skim milk powder
in PBS (pH 7.4)).
Incubation with Primary Antibody:
For 4 cm2 membranes, we recommend using a final volume of 1 ml of diluted antibody solution. To obtain
comparable results for a 20 cm2 membrane, use 5 mls of the diluted antibody solution.
4. Rinse membranes with 5% Blotto. Dilute the antibody (at the appropriate dilution) in 5% Blotto. Incubate for 1
hour at 37°C, 2 hours at RT (room temperature) or overnight at 4°C with agitation.
5. Decant antibody solution. Wash the membrane 5 times for 5-10 minutes at RT in 1% Blotto (1% dry, non-fat skim
milk powder in PBS (pH 7.4)). Washing should be performed with vigorous agitation over a minimum 30-minute
period.
Incubation with Secondary Antibody
6. Incubate membranes with secondary HRP conjugate diluted in 5% Blotto for 1 hour at RT with gentle shaking.
The dilution of the secondary antibody conjugate will vary according to manufacturer's specifications; however,
we recommend using a 1:2000-1:5000 dilution of conjugated antibody.
7. Repeat step 5.
8. Wash membrane with PBS (pH 7.4) for 5 minutes with agitation before proceeding to the chemiluminescence
reaction.
Chemiluminescence Reaction
9. Prepare and use the chemiluminescent substrate according to the manufacturer's instructions.
10. Immediately wrap membrane and expose to X-ray film for 15 seconds to a 1-hour period. The time exposure may
vary according to the amount of antigen and should be optimized for each source. Please note that the background
can increase with time exposure.
Other information
References
1. Dignam, J.D., et al., (1983), Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei,
Nucleic Acids Res., 11(5), 1475-1489.
2. Towbin, H., et al., (1979), Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some
applications, PNAS, 76(9), 4350-43
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interbiotech@interchim.com
P.2
FT-Q99980
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P.3