Ms X PCNA

Species Reactivity	Key Applications	Host	Format	Antibody Type
B, Ca, H, M, R, Rb, F, Vrt	IF, ICC, IH(P)	M	Ascites	Monoclonal Antibody

Species Reactivity

Key Applications

Host

Format

Antibody Type

B, Ca, H, M, R, Rb, F, Vrt

IF, ICC, IH(P)

Ascites

Monoclonal Antibody

Description
Catalogue Number	MAB4078
Brand Family	Chemicon®
Trade Name	Chemicon
Description	Anti-PCNA Antibody, clone 131-11912
Alternate Names	Proliferating Cell Nuclear Antigen DNA Polymerase delta Processivity Factor

Description

Catalogue Number

MAB4078

Brand Family

Chemicon®

Trade Name

Chemicon

Description

Anti-PCNA Antibody, clone 131-11912

Alternate Names

Proliferating Cell Nuclear Antigen
DNA Polymerase delta Processivity Factor

Product Information
Format	Ascites
Presentation	Ascites fluid containing no preservatives

Product Information

Format

Ascites

Presentation

Ascites fluid containing no preservatives

Applications
Application	Anti-PCNA Antibody, clone 131-11912 is a mouse monoclonal antibody for detection of PCNA also known as Proliferating Cell Nuclear Antigen, DNA Polymerase delta Processivity Factor & has been validated in IF, ICC,IHC(P).
Key Applications	Immunofluorescence Immunocytochemistry Immunohistochemistry (Paraffin)
Application Notes	Immunohistochemistry on formalin fixed, deparaffinized tissue sections Frozen tissue sections. Smears. Cytospins. * Do not use Serum in blocking steps. Also see http://ajp.amjpathol.org/cgi/content/abstract/141/6/1453 for important reactivity information on 19A2 in paraffin fixed tissues. Final working dilutions must be determined by end user. IMMUNOHISTOCHEMICAL PROCEDURE FOR MAB4078 The following histochemical immunoperoxidase protocol has been optimized for use with MAB4078. METHODOLOGY: This method is used with standard streptavidin or ABC immunoperoxidase methods using a DAB substrate run at room temperature with paraffin or frozen sections, smears or cytospins. NOTE: Optimal fixation time for paraffin processed tissue fixed in 10% Neutral Buffered Formalin is 12-14 hours. REQUIRED SUPPLEMENTAL MATERIAL: 1. 3% Hydrogen Peroxide: commercially available. 2. PBS 3. Ethyl Alcohol: histology grade or equivalent 4. Methanol: histology grade or equivalent 5. Acetone: histology grade or equivalent 6. Xylene (histology grade) or Xylene Substitute (Hemo-D) 7. Mounting Media: Krystalon (Harleco) or equivalent 8. Gill #1 Hematoxylin: Sigma #GHS-1-32 or equivalent 9. Goat Anti-Mouse Biotin 10. Streptavidin HRP (optional reagent) 11. 3,3'Diaminobenzidine tetrahydrochloride (DAB): Sigma #D-5637 or equivalent 12. ABC: Vector Kit #PK4000 or equivalent REAGENT PREPARATION: 1. Primary antibody: Prepare a stock solution of antibody in PBS 2. Goat Anti-Mouse Biotin: Approximate dilution of 1:100 in PBS (10 mg/test) 3. Streptavidin: 5 mL stock solution and 995 mL PBS (1/200 dilution). Let sit 15 minutes before use (yield as a volume sufficient for ~20 slides) OR if using ABC kit from Vector follow as instructed. 4. DAB: Reconstitute 1.5 mg of powder with 100 mL of PBS. Activate 15 minutes prior to use by adding 100 mL 3% H2O2 (yields a volume sufficient for ~20 slides). WARNING: DAB is a possible carcinogen. Avoid contact with skin. 5. PBS: Using a graduated cylinder, reconstitute with 500 mL of distilled water. 6. Alcohol Baths: All dilutions are alcohols made with distilled water. PROCEDURE: PARAFFIN SECTIONS (cut at 4-5 microns, 10% Neutral Buffered Formalin fixed): 1. Place slides in 62°C oven for 15 minutes. 2. Place slides in Hemo-D, or xylene, for 15 minutes. 3. De-paraffinize slides through baths of: A) xylene/100% ethanol 1:1 (or Hemo-D/100% ethanol 1:1), B) 100% ethyl alcohol x2, C) 95% ethyl alcohol, D) 90% ethyl alcohol, E) 80% ethyl alcohol, F) 70% ethyl alcohol x2 (2 minutes each bath). 4. Place slides in running cool tap water for 5 minutes. 5. Shake off excess water. Place slides on incubation racks held at room temperature. Circle sections with PAP pen (Newcomer, Inc.) or diamond pen to isolate section. 6. Flood sections with primary antibody and incubate for 35 - 40 minutes. 7. Wipe off excess PBS. Flood sections with Goat Anti-Mouse Biotin reagent and incubate for 35-40 minutes. 8. Wash with PBS 3-5 min 9. Wash again with PBS 3-5 min. 10. Wipe off excess PBS. Flood sections with streptavidin-HRP and incubate for 10 minutes OR flood sections with ABC and incubate for 35 - 40 minutes. 11. Wash with PBS 3-5 min. 12. Wipe off excess PBS. Flood sections with DAB and incubate for 6-8 minutes. 13. Place slides in cool running tap water for 5 minutes. 14. Place slides in Gills #1 hematoxylin for 90 seconds. 15. Place slides in cool running tap water for 5 minutes. 16. Dehydrate slides through baths of A) 70% ethyl alcohol, B) 80% ethyl alcohol, C) 90% ethyl alcohol, D) 95% ethyl alcohol x2, E) 100% ethyl alcohol x2, F) xylene/100% ethanol 1:1 (or Hemo-D/100% ethanol 1:1) x2, G) xylene or Hemo-D x2 (2 minutes each bath. 17. Mount slides using mounting media. FROZEN SECTIONS (cut at 8 microns): Pull sections from freezer (or cut fresh), air dry at room temperature (or under a fan for 15 minutes), fixed in 4ºC acetone for 4 minutes, air dry for 30 minutes at room temperature (or under a fan for 15 minutes), then follow above procedure beginning at #5. SMEARS: Methanol fix for 30 minutes at 4ºC, then follow above procedure beginning at #5. CYTOSPINS: Cell lines and other cell populations prepared by resuspending, in each well, ~40,000 cells using 500 mL 1% Normal Goat Serum per well. Centrifuge for 6 minutes at 1200 RPM. Air dry for 30 minutes at room temperature (or under a fan for 15 minutes). If tumor cells are being screened, fix in RT acetone for 2 - 3 minutes. If peripheral blood preps are being screened, fix in 100% methanol for 20 minutes. Air dry the slides for 30 minutes at room temperature (or 15 minutes under a fan) and begin at #5 above. NOTE: When staining using the PCNA antibodies as the primary DO NOT use Normal Goat Serum or any other serum blocking step.

Applications

Application

Anti-PCNA Antibody, clone 131-11912 is a mouse monoclonal antibody for detection of PCNA also known as Proliferating Cell Nuclear Antigen, DNA Polymerase delta Processivity Factor & has been validated in IF, ICC,IHC(P).

Key Applications

Immunofluorescence
Immunocytochemistry
Immunohistochemistry (Paraffin)

Application Notes

Immunohistochemistry on formalin fixed, deparaffinized tissue sections
Frozen tissue sections.
Smears.
Cytospins.
* Do not use Serum in blocking steps.

Also see http://ajp.amjpathol.org/cgi/content/abstract/141/6/1453 for important reactivity information on 19A2 in paraffin fixed tissues.

Final working dilutions must be determined by end user.

IMMUNOHISTOCHEMICAL PROCEDURE FOR MAB4078

The following histochemical immunoperoxidase protocol has been optimized for use with MAB4078.

METHODOLOGY: This method is used with standard streptavidin or ABC immunoperoxidase methods using a DAB substrate run at room temperature with paraffin or frozen sections, smears or cytospins.

NOTE: Optimal fixation time for paraffin processed tissue fixed in 10% Neutral Buffered Formalin is 12-14 hours.

REQUIRED SUPPLEMENTAL MATERIAL:

1. 3% Hydrogen Peroxide: commercially available.

2. PBS

3. Ethyl Alcohol: histology grade or equivalent

4. Methanol: histology grade or equivalent

5. Acetone: histology grade or equivalent

6. Xylene (histology grade) or Xylene Substitute (Hemo-D)

7. Mounting Media: Krystalon (Harleco) or equivalent

8. Gill #1 Hematoxylin: Sigma #GHS-1-32 or equivalent

9. Goat Anti-Mouse Biotin

10. Streptavidin HRP (optional reagent)

11. 3,3'Diaminobenzidine tetrahydrochloride (DAB): Sigma #D-5637 or equivalent

12. ABC: Vector Kit #PK4000 or equivalent

REAGENT PREPARATION:

1. Primary antibody: Prepare a stock solution of antibody in PBS

2. Goat Anti-Mouse Biotin: Approximate dilution of 1:100 in PBS (10 mg/test)

3. Streptavidin: 5 mL stock solution and 995 mL PBS (1/200 dilution). Let sit 15 minutes before use (yield as a volume sufficient for ~20 slides) OR if using ABC kit from Vector follow as instructed.

4. DAB: Reconstitute 1.5 mg of powder with 100 mL of PBS. Activate 15 minutes prior to use by adding 100 mL 3% H2O2 (yields a volume sufficient for ~20 slides). WARNING: DAB is a possible carcinogen. Avoid contact with skin.

5. PBS: Using a graduated cylinder, reconstitute with 500 mL of distilled water.

6. Alcohol Baths: All dilutions are alcohols made with distilled water.

PROCEDURE:

PARAFFIN SECTIONS

(cut at 4-5 microns, 10% Neutral Buffered Formalin fixed):

1. Place slides in 62°C oven for 15 minutes.

2. Place slides in Hemo-D, or xylene, for 15 minutes.

3. De-paraffinize slides through baths of: A) xylene/100% ethanol 1:1 (or Hemo-D/100% ethanol 1:1), B) 100% ethyl alcohol x2, C) 95% ethyl alcohol, D) 90% ethyl alcohol, E) 80% ethyl alcohol, F) 70% ethyl alcohol x2 (2 minutes each bath).

4. Place slides in running cool tap water for 5 minutes.

5. Shake off excess water. Place slides on incubation racks held at room temperature. Circle sections with PAP pen (Newcomer, Inc.) or diamond pen to isolate section.

6. Flood sections with primary antibody and incubate for 35 - 40 minutes.

7. Wipe off excess PBS. Flood sections with Goat Anti-Mouse Biotin reagent and incubate for 35-40 minutes.

8. Wash with PBS 3-5 min

9. Wash again with PBS 3-5 min.

10. Wipe off excess PBS. Flood sections with streptavidin-HRP and incubate for 10 minutes OR flood sections with ABC and incubate for 35 - 40 minutes.

11. Wash with PBS 3-5 min.

12. Wipe off excess PBS. Flood sections with DAB and incubate for 6-8 minutes.

13. Place slides in cool running tap water for 5 minutes.

14. Place slides in Gills #1 hematoxylin for 90 seconds.

15. Place slides in cool running tap water for 5 minutes.

16. Dehydrate slides through baths of A) 70% ethyl alcohol, B) 80% ethyl alcohol, C) 90% ethyl alcohol, D) 95% ethyl alcohol x2, E) 100% ethyl alcohol x2, F) xylene/100% ethanol 1:1 (or Hemo-D/100% ethanol 1:1) x2, G) xylene or Hemo-D x2 (2 minutes each bath.

17. Mount slides using mounting media.

FROZEN SECTIONS (cut at 8 microns): Pull sections from freezer (or cut fresh), air dry at room temperature (or under a fan for 15 minutes), fixed in 4ºC acetone for 4 minutes, air dry for 30 minutes at room temperature (or under a fan for 15 minutes), then follow above procedure beginning at #5.

SMEARS: Methanol fix for 30 minutes at 4ºC, then follow above procedure beginning at #5.

CYTOSPINS: Cell lines and other cell populations prepared by resuspending, in each well, ~40,000 cells using 500 mL 1% Normal Goat Serum per well. Centrifuge for 6 minutes at 1200 RPM. Air dry for 30 minutes at room temperature (or under a fan for 15 minutes). If tumor cells are being screened, fix in RT acetone for 2 - 3 minutes. If peripheral blood preps are being screened, fix in 100% methanol for 20 minutes. Air dry the slides for 30 minutes at room temperature (or 15 minutes under a fan) and begin at #5 above.

NOTE: When staining using the PCNA antibodies as the primary DO NOT use Normal Goat Serum or any other serum blocking step.

Biological Information
Immunogen	Purified PCNA
Clone	131-11912
Host	Mouse
Specificity	Specific for PCNA a 36 kDa proliferation associated antigen.
Isotype	IgM
Species Reactivity	Bovine Canine Human Mouse Rat Rabbit Fish Vertebrates
Antibody Type	Monoclonal Antibody
Entrez Gene Number	NM_002592.2 NM_182649.1
Entrez Gene Summary	The protein encoded by this gene is found in the nucleus and is a cofactor of DNA polymerase delta. The encoded protein acts as a homotrimer and helps increase the processivity of leading strand synthesis during DNA replication. In response to DNA damage, this protein is ubiquitinated and is involved in the RAD6-dependent DNA repair pathway. Two transcript variants encoding the same protein have been found for this gene. Pseudogenes of this gene have been described on chromosome 4 and on the X chromosome.
Gene Symbol	PCNA Cyclin cyclin MGC8367
UniProt Number	P12004
UniProt Summary	FUNCTION: SwissProt: P12004 # This protein is an auxiliary protein of DNA polymerase delta and is involved in the control of eukaryotic DNA replication by increasing the polymerase's processibility during elongation of the leading strand. SIZE: 261 amino acids; 28769 Da SUBUNIT: Homotrimer. Interacts with KCTD10 (By similarity). Forms a complex with activator 1 heteropentamer in the presence of ATP. Interacts with POLH, POLK, DNMT1, ERCC5/XPG, FEN1, CDC6, APEX2 and POLDIP2. Interacts with EXO1 and SHPRH. SUBCELLULAR LOCATION: Nucleus. PTM: Upon methyl methanesulfonate-induced DNA damage, mono- ubiquitinated by the UBE2B-RAD18 complex on Lys-164. This induces non-canonical poly-ubiquitination on Lys-164 through 'Lys-63' linkage of ubiquitin moieties by the E2 complex UBE2N-UBE2V2 and the E3 ligase SHPRH, whih is required for DNA repair. DISEASE:SwissProt: P12004 # Antibodies are present in sera from patients with systemic lupus erythematosus. SIMILARITY: SwissProt: P12004 ## Belongs to the PCNA family.

Biological Information

Immunogen

Purified PCNA

Clone

131-11912

Host

Mouse

Specificity

Specific for PCNA a 36 kDa proliferation associated antigen.

Isotype

IgM

Species Reactivity

Bovine
Canine
Human
Mouse
Rat
Rabbit
Fish
Vertebrates

Antibody Type

Monoclonal Antibody

Entrez Gene Number

Entrez Gene Summary

The protein encoded by this gene is found in the nucleus and is a cofactor of DNA polymerase delta. The encoded protein acts as a homotrimer and helps increase the processivity of leading strand synthesis during DNA replication. In response to DNA damage, this protein is ubiquitinated and is involved in the RAD6-dependent DNA repair pathway. Two transcript variants encoding the same protein have been found for this gene. Pseudogenes of this gene have been described on chromosome 4 and on the X chromosome.

Gene Symbol

PCNA
Cyclin
cyclin
MGC8367

UniProt Number

P12004

UniProt Summary

FUNCTION: SwissProt: P12004 # This protein is an auxiliary protein of DNA polymerase delta and is involved in the control of eukaryotic DNA replication by increasing the polymerase's processibility during elongation of the leading strand.
SIZE: 261 amino acids; 28769 Da
SUBUNIT: Homotrimer. Interacts with KCTD10 (By similarity). Forms a complex with activator 1 heteropentamer in the presence of ATP. Interacts with POLH, POLK, DNMT1, ERCC5/XPG, FEN1, CDC6, APEX2 and POLDIP2. Interacts with EXO1 and SHPRH.
SUBCELLULAR LOCATION: Nucleus.
PTM: Upon methyl methanesulfonate-induced DNA damage, mono- ubiquitinated by the UBE2B-RAD18 complex on Lys-164. This induces non-canonical poly-ubiquitination on Lys-164 through 'Lys-63' linkage of ubiquitin moieties by the E2 complex UBE2N-UBE2V2 and the E3 ligase SHPRH, whih is required for DNA repair.
DISEASE:SwissProt: P12004 # Antibodies are present in sera from patients with systemic lupus erythematosus.
SIMILARITY: SwissProt: P12004 ## Belongs to the PCNA family.

Product Usage Statements
Usage Statement	Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Product Usage Statements

Usage Statement

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Storage and Shipping Information
Storage Conditions	Maintain frozen at -20°C in undiluted aliquots for up to 12 months from date of receipt. Avoid repeated freeze/thaw cycles.

Storage and Shipping Information

Storage Conditions

Maintain frozen at -20°C in undiluted aliquots for up to 12 months from date of receipt. Avoid repeated freeze/thaw cycles.

Packaging Information
Material Size	100 µL

Packaging Information

Material Size

100 µL

实验耗材

分子生物学试剂

蛋白与免疫学

生化试剂

细胞及检测试剂

色谱及层析

仪器设备

实验服务

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