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Lamin A/C monoclonal antibody (4C11)

 
ALX-804-672-C200 200 µg 430.00 USD
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Product Specification

Clone:4C11
 
Host:Mouse
 
Isotype:IgG2a
 
Immunogen:Recombinant human lamin A/C (aa 430-545).
 
UniProt ID:P02545
 
Species reactivity:Human, Mouse, Rat
Hamster, Monkey
 
Crossreactivity:Does not cross-react with lamin B1 and lamin B2.
 
Applications:ICC, IP, WB
 
Recommended Dilutions/Conditions:Immunocytochemistry (1:100)
Immunoprecipitation (5-10µl/assay)
Western Blot (1:2,000-1:5,000)
Suggested dilutions/conditions may not be available for all applications.
Optimal conditions must be determined individually for each application.
 
Formulation:Liquid. Hybridoma supernatant containing sodium azide.
 
Handling:Avoid freeze/thaw cycles.
 
Shipping:Shipped on Blue Ice
 
Short Term Storage:+4°C
 
Long Term Storage:-20°C
 
Scientific Background:Lamin A is a type V intermediate filament protein, involved in maintaining nuclear shape and structure, transcriptional regulation, nuclear pore positioning and function, and heterochromatin organization. Mutations in lamin A are associated with several genetic degenerative disease, including Hutchinson-Gilford progeria syndrome (HGPS), which is caused by Progerin a truncated version of lamin A. A-type lamins have been described prognostic biomarkers for colorectal cancer, and lamin A (but not lamin C) is a potential adult stem cell biomarker in colonic crypts.
 
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Figure 1: Western blot analysis of lamin A/C using MAb to Lamin A/C (4C11) (Prod. No. ALX-804-672). Method: HeLa cells were lysed with 1 x GSD boiling buffer. The cell lysate was electrophoretically separated on a 7.5% SDS-PAG and transferred onto a nitrocellulose membrane. One lane contains approximately 7.5µg of whole cell protein. The nitrocellulose membrane was blocked for 1 h with 3% NFDM/PBS-T and then incubated with MAb to Lamin A/C (4C11) at the indicated dilutions (diluted in 0.5% NFDM/PBS-T) for 2 h at RT. The membrane was washed with PBS-T and then incubated for 1 h with HRP coupled anti-mouse antibody (1:5000). The membrane was washed 3 x 10 min with PBS-T and then incubated for 1 min with ECL solution. The signal was detected on a HR-HA 30 X-ray film. Exposure time was 3 min.
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Figure 2: Immunoprecipitation of lamin A/C using MAb to Lamin A/C (4C11) (Prod. No. ALX-804-672).Method: For each IP 2mg of whole cell lysate from HeLa cells ectopically expressing FLAG-tagged lamin A were used. The lysates were incubated with the indicated volumes of hybridoma supernatant  for 1 hour at 4°C. Then 30µl of Protein A + G sepharose (1:1 suspension) were added and incubated for 1 hour at 4°C. Beads were washed, boiled in 30µl 1x GSD boiling buffer for 5 min, and the proteins were loaded on a 10% SDS-PAG and electrophoretically separated (2% of lysate before and after the IP were also loaded). The proteins were blotted on a nitrocellulose membrane. The membrane was blocked with 3% NFDM-PBS/T for 1 hour and then incubated with MAb to Lamin A/C (4C11) (1:1000 in 0.5% NFDM-PBS/T) over night, 4°C. The membrane was washed 3 x 10 min with PBS/T and then incubated with anti-mouse HRP (1:5000 in 0.5% NFDM-PBS/T) for 1 hour at RT. The membrane was washed 3 x 10 min and then incubated for 1 min with ECL solution. The signal was detected on a HR-HA 30 X-ray film. Exposure time was 5 sec.
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Figure 3: Immunocytochemistry using MAb to Lamin A/C (4C11) (Prod. No. ALX-804-672).Method: HeLa cells ectopically expressing FLAG-tagged lamin A were grown on coverslips and fixed with ice cold methanol for 10 min at 4°C, permeabilized with 0.5% Triton-X/PBS for 10 min at RT, blocked with 0.2% gelatine/PBS for 1h and then incubated with MAb to Lamin A/C (4C11) (1:100 in 0.2% gelatine/PBS) for 1 h at RT. Afterwards the cells were incubated with anti-mouse IgG Alexa Fluor 594 antibody (1:500). DNA was counterstained with Hoechst 33342. Pictures were taken with a confocal microscope.
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Figure 4: Western blot analysis of lamin A/C in different species using MAb to Lamin A/C (4C11) (Prod. No. ALX-804-672).Method: GSD-lysates from different species were electrophoretically separated on a 7.5% SDS PAG and blotted on a nitrocellulose membrane. The membrane was blocked with 3% NFDM-PBS/T for 1 hour and incubated with MAb to Lamin A/C (4C11) 1:1000 in 0.5% NFDM-PBS/T o/N at 4°C (as loading control anti β-actin, 1:5000). The membrane was washed 3 x 10 min with PBS/T and incubated with anti-mouse HRP, 1:5000 in 0.5% NFDM-PBS/T 1 h at RT. The membrane was washed 3 x 10 min with PBS/T and then incubated for 1 min with ECL solution. The signal was detected on a HR-HA 30 X-ray film. Exposure time was 10 sec.
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Figure 5:Lamin A/C, mAb (4C11) (Prod. No. ALX-804-672) does not cross-react with lamin B1 or B2.
Method:Western blot analysis of bacterially expressed fusion proteins of GST with the lamin A/C Ig-fold domain (aa 432-544), the lamin B1 Ig-fold domain (aa 434-547), or the lamin B2 Ig-fold domain (aa 466-580). Incubation with a GST-specific antibody (left blot) ensured equal loading. Incubation with the lamin A/C antibody, clone 4C11 (right blot), demonstrates that it does not cross-react with lamin B1 or B2 (even when the film was heavily overexposed as shown in figure).
Calculated size of GST-fusion proteins is ~40kDa. The prominent band at ~25kDa represents a degradation product of GST-Lamin A/C-Igfold.
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Product Literature References

Anti-RAINBOW dye-specific antibodies as universal tools for the visualization of prestained protein molecular weight markers in Western blot analysis: S. Schüchner, et al.; Sci. Rep. 6, 31363 (2016), Application(s): Western blotting/Dot blotting, Abstract; Full Text

General Literature References

Lamin A-dependent misregulation of adult stem cells associated with accelerated ageing: P. Scaffidi & T. Misteli; Nat. Cell Biol. 10, 452 (2008), Abstract; Full Text
Lamin A/C is a risk biomarker in colorectal cancer: N.D. Willis, et al.; PLoS ONE 3, e2988 (2008), Abstract; Full Text
Lamin A/C, laminopathies and premature ageing: B. Liu & Z. Zhou; Histol. Histopathol. 23, 747 (2008), Abstract;
The mutant form of lamin A that causes Hutchinson-Gilford progeria is a biomarker of cellular aging in human skin: D. McClintock, et al.; PLoS ONE 2, e1269 (2007), Abstract; Full Text

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