Overview

  • Product name
  • Description
    Mouse monoclonal to XPD
  • Host species
    Mouse
  • Tested applications
    Suitable for: WB, Flow Cyt, IPmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Recombinant full length protein, corresponding to amino acids 1-406 of Human XPD

Properties

Applications

Our Abpromise guarantee covers the use of ab54676 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB Use a concentration of 1 - 5 µg/ml. Predicted molecular weight: 87 kDa.
Flow Cyt Use 1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

IP Use at an assay dependent concentration.

Target

  • Function
    ATP-dependent 5'-3' DNA helicase, component of the core-TFIIH basal transcription factor. Involved in nucleotide excision repair (NER) of DNA by opening DNA around the damage, and in RNA transcription by RNA polymerase II by anchoring the CDK-activating kinase (CAK) complex, composed of CDK7, cyclin H and MAT1, to the core-TFIIH complex. Involved in the regulation of vitamin-D receptor activity. As part of the mitotic spindle-associated MMXD complex it plays a role in chromosome segregation. Might have a role in aging process and could play a causative role in the generation of skin cancers.
  • Involvement in disease
    Defects in ERCC2 are the cause of xeroderma pigmentosum complementation group D (XP-D) [MIM:278730]; also known as XP group D (XPD). Xeroderma pigmentosum is an autosomal recessive pigmentary skin disorder characterized by solar hypersensitivity of the skin, high predisposition for developing cancers on areas exposed to sunlight and, in some cases, neurological abnormalities. Some XP-D patients present features of Cockayne syndrome, including dwarfism, sensorineural deafness, microcephaly, mental retardation, pigmentary retinopathy, ataxia, decreased nerve conduction velocities.
    Defects in ERCC2 are a cause of trichothiodystrophy photosensitive (TTDP) [MIM:601675]. TTDP is an autosomal recessive disease characterized by sulfur-deficient brittle hair and nails, ichthyosis, mental retardation, impaired sexual development, abnormal facies and cutaneous photosensitivity correlated with a nucleotide excision repair (NER) defect. Neonates with trichothiodystrophy and ichthyosis are usually born with a collodion membrane. The severity of the ichthyosis after the membrane is shed is variable, ranging from a mild to severe lamellar ichthyotic phenotype. There are no reports of skin cancer associated with TTDP.
    Defects in ERCC2 are the cause of cerebro-oculo-facio-skeletal syndrome type 2 (COFS2) [MIM:610756]. COFS is a degenerative autosomal recessive disorder of prenatal onset affecting the brain, eye and spinal cord. After birth, it leads to brain atrophy, hypoplasia of the corpus callosum, hypotonia, cataracts, microcornea, optic atrophy, progressive joint contractures and growth failure. Facial dysmorphism is a constant feature. Abnormalities of the skull, eyes, limbs, heart and kidney also occur.
  • Sequence similarities
    Belongs to the helicase family. RAD3/XPD subfamily.
    Contains 1 helicase ATP-binding domain.
  • Post-translational
    modifications
    ISGylated.
  • Cellular localization
    Nucleus. Cytoplasm > cytoskeleton > spindle.
  • Information by UniProt
  • Database links
  • Alternative names
    • TFIIH 80 kDa subunit antibody
    • Basic transcription factor 2 80 kDa subunit antibody
    • BTF2 p80 antibody
    • COFS 2 antibody
    • COFS2 antibody
    • CXPD antibody
    • DNA excision repair protein ERCC 2 antibody
    • DNA excision repair protein ERCC-2 antibody
    • DNA repair protein complementing XP D cells antibody
    • DNA repair protein complementing XP-D cells antibody
    • EM9 antibody
    • ERCC 2 antibody
    • ERCC2 antibody
    • ERCC2_HUMAN antibody
    • Excision repair 2 antibody
    • Excision repair cross complementing rodent repair deficiency complementation antibody
    • Excision repair cross complementing rodent repair deficiency, complementation group 2 antibody
    • MAG antibody
    • MGC102762 antibody
    • MGC126218 antibody
    • MGC126219 antibody
    • OTTHUMP00000045860 antibody
    • OTTHUMP00000045861 antibody
    • OTTHUMP00000045862 antibody
    • OTTHUMP00000045863 antibody
    • TFIIH 80 kDa subunit antibody
    • TFIIH basal transcription factor complex 80 kDa subunit antibody
    • TFIIH Basal Transcription Factor Complex Helicase Subunit antibody
    • TFIIH basal transcription factor complex helicase XPD subunit antibody
    • TFIIH basal transcription factor complex p80 subunit antibody
    • TFIIH p80 antibody
    • TTD antibody
    • Xeroderma pigmentosum complementary group D antibody
    • Xeroderma pigmentosum group D complementing protein antibody
    • Xeroderma pigmentosum group D-complementing protein antibody
    • XPD antibody
    • XPDC antibody
    see all

Images

  • XPD antibody (ab54676) at 1ug/lane + HeLa cell lysate at 25ug/lane.
  • XPD was immunoprecipitated using 0.5mg Hela whole cell extract, 10µg of Mouse monoclonal to XPD and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
    The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab54676.
    Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/5000 dilution.
    Band: 150kDa: SMC1; Non specific - 41 and 42kDa: We are unsure as to the identity of this extra band.
  • Overlay histogram showing HeLa cells stained with ab54676 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab54676, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

References

This product has been referenced in:
  • Odermatt DC & Gari K The CIA Targeting Complex Is Highly Regulated and Provides Two Distinct Binding Sites for Client Iron-Sulfur Proteins. Cell Rep 18:1434-1443 (2017). Read more (PubMed: 28178521) »
  • Servant G  et al. The Nucleotide Excision Repair Pathway Limits L1 Retrotransposition. Genetics 205:139-153 (2017). Read more (PubMed: 28049704) »
See all 10 Publications for this product

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

Answer

Vielen Dank für Ihre Antworten.

Ich möchte noch eine Anmerkung bezüglich der Positivkontrolle im Falle des ab54676und ab96089machen: Laut UniGene expremierencolorektale Tumore nicht besonders viel von der entsprechenden RNA. Für den ab54676würde ich daher eher eine Lymphoma Zelllinie wie Daudi empfehlen. Da ich leider im Falle des ab96089keine Alternative empfehlen kann, möchte ich vorschlagen, dass Sie die Ladung auf 50 ug erhöhen.


Ich hoffe, dies hilft Ihnen weiter. Bitte zögern Sie nicht, sich wieder bei uns zu melden, falls sich Ihre Ergebnisse nicht verbessern.

Benutzen Sie unsere Produkte? Schicken Sie uns einen Abreview. Verdienen Sie sich eine Belohnung!
https://www.abcam.com/abreviews

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Question

Anbei der vollständig ausgefüllte Fragebogen!



Vielen dank!

Mit besten grüßen

1) Abcam product code ab 96089, ab 5063, ab54676, ab54645, ab 6264

2) Abcam order reference number or product batch number

3) Description of the problem: see pictures below, either to high cross reactivity or no signal detectable

4) Sample preparation:
Type of sample (whole cell lysates, fraction, recombinant protein…): whole cell lysate
Lysis buffer: RIPA
Protease inhibitors: sigma (http://www.sigmaaldrich.com/catalog/product/sigma/p8340?lang=de®ion=AT)
Phosphatase inhibitors: sigma
Reducing agent
Boiling for ≥5 min? yes/no yes
Protein loaded ug/lane or cells/lane 15-100 µg
Positive control yes
Negative control

5) Percentage of gel 10%
Type of membrane Nylon
Protein transfer verified yes (Ponseau red)
Blocking agent and concentration 5% BSA in 1xTBS/T (as well as 5% milk in 1xTBS/T but in milk no signal could be detected)
Blocking time 4 °C over night, as well as 1 h room temperature
Blocking temperature see above

6) Primary antibody (If more than one was used, describe in “additional notes”) :
Concentration or dilution: FancG 1:500, FancA 1:1000, XPD 1:1000, CSB 1:1500
Diluent buffer 5% BSA as well as milk in 1xTBS/T
Incubation time over night
Incubation temperature: 4°C

7) Secondary antibody:
Species:
Reacts against:
Concentration or dilution 1:1000
Diluent buffer 5% BSA in 1x TBS/T
Incubation time: 1h
Incubation temperature: room temperature
Fluorochrome or enzyme conjugate: HRP

8) Washing after primary and secondary antibodies:
Buffer 1x TBS/T
Number of washes 3

9)Detection method
10) How many times have you run this staining?
Do you obtain the same results every time?
What steps have you altered to try and optimize the use of this antibody?
* changed incubation time
* changes milk to BSA
* blocked over night or 1h

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Answer

Vielen Dank für Ihren Anruf und dafür, dass Sie sich die Zeit genommen haben, unseren Fragebogen auszufüllen.

Es tut mir leid, dass Sie Probleme mit unseren Antikörper hatten.

Um Ihr Problem zu lösen, möchte ich die folgenden Vorschläge zu Veränderung Ihres Protokolls machen, bzw noch ein paar zusätzliche Fragen stellen:

1. Um was für Zelllinien handelt es sich hier, bzw aus welcher Spezies wurden die Zellen ursprünglich gewonnen? Sind die Zellen transfektiert?

2. Was ist Ihre Positiv- Kontrolle?

3.Verwenden Sie einen Anti-Mouse Sekundär- AKim falle der ab54645 und ab54676?

4. Verwenden Sie immer einen frischen Blot pro Antikörper oder strippen Sie diesen zwischendurch?

5. Haben Sie Ihre Zellen transfektiert oder wollen Sie das endogene Protein detektieren? Für den ersten Fall senken Sie den Proteingehalt auf 5- 10ug pro Bahn ab, im zweiten Fall gehen Sie runter auf 20 ug.

6. Filtern Sie die Blockmittelllösung, dadurch sollten die schwarzen Punkte verschwinden und die Ergebnisse klarer werden.

7. Senken Sie die Konzentration der Sekundär- Antikörper auf mindestens 1/5000 ab.

8. Blockieren Sie weiterhin eine Stunde und inkubieren sie in der höchsten von uns empfohlenenVerdünnung über Nacht bei 4C.



Bitte lassen Sie mich wissen, ob ich Ihnen helfen konnte und zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.

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Answer

Vielen Dank für Ihren Anruf.

Es tut mir leid zu hören, dass Sie Probleme mit diesen Antikörpern haben.

Ich habe unseren Fragebogen als Word-Dokument an diese E-Mail angehängt. Durch das Ausfüllen des Fragebogens erhalten wir alle nötigen Informationen über Ihre Proben und Ihr Protokoll. Sobald Sie dieses Formular an uns zurückgeschickt haben, werden wir uns Ihr Protokoll ansehen und möglichst Veränderungsvorschläge machen, die Ihre Ergebnisse verbessern werden. Falls sich herausstellt, dass der Antikörper nicht so funktioniert, wie auf dem Datenblatt beschrieben und er innerhalb der letzten 180 Tage gekauft wurde, werden wir Ihnen gerne einen Ersatz oder eine Gutschrift schicken.


Ich freue mich, bald wieder von Ihnen zu hören.

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - nuclear (fibroblast cell line)
Loading amount
100 µg
Specification
fibroblast cell line
Gel Running Conditions
Reduced Denaturing (6 %)
Blocking step
Blocking buffer from another company as blocking agent as blocking agent for 30 minute(s) · Concentration: 5µg/mL · Temperature: 21°C

Mrs. Annika Schäfer

Verified customer

Submitted Feb 10 2009

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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