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  • Immunofluorescence analysis of paraformaldehyde fixed HeLa cells,  permeabilized with 0.15% Triton. Primary incubation 1hr (1:100 dilution) followed by Alexa Fluor® 488 secondary antibody (1:1000 dilution), showing nuclear and cytoplasmic staining. The nuclear stain is DAPI (blue). Negative control: Mouse IgG1 negative control followed by Alexa Fluor® 488 secondary antibody.
  • (0.1µg/ml) staining in HeLa(A), (0.01µg/ml) staining in HeLa-nuclear(B), (0.1µg/ml) staining in NIH3T3(C) and (0.03µg/ml) staining in NIH3T3-nuclear(D) cells lysate (35µg protein in RIPA buffer). Primary incubation was 1 hour. Detected by chemiluminescence.
  • Histone H3 (tri methyl K9) Monoclonal Antibody [6F12-H4]
  • ChIP using 1ug chromatin and 2ug Anti H3K9me3 Antibody (IQ549) clone 6F12-H4

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Histone H3 (tri methyl K9) Monoclonal Antibody [6F12-H4]

Catalogue Number: IQ549

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  • £328.90

Datasheet

Primary Description: Mouse Anti-Histone H3 (tri methyl K9) [6F12-H4]
Target Antigen: Histone H3 (tri methyl K9)
Catalogue Number: IQ549
Quantity: 0.1mg
Concentration: 1mg/ml
Clone: 6F12-H4
Host: Mouse
Isotype: IgG1
Myeloma/Fusion Partners: X63-Ag8.653
Immunogen: Synthetic peptide of Human Histone H3, tri methylated at K9
Species Reactivity: Reacts with Human and cow. predicted to detect many other species due to sequence identity such as Mouse, Rat, Rabbit, Hamster, Pig, Saccharomyces cerevisiae, Fruit fly (Drosophila melanogaster), Non Human Primates
Purification: Protein G
Format: Purified antibody (from supernatant) containing PBS + 0.1% sodium azide
Applications: Dot blot, ELISA, WB, ChIP, IHC-Fr, ICC/IF
Dilutions: Optimal antibody dilution should be determined by titration, however as a guideline try up to 1:1000 for western blot. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).Can be blocked with Histone H3 peptide - tri methyl K9 
Also Known As: Histone H3.1 antibody;FLJ92264 antibody;H3 histone antibody;H3 histone family, member A antibody;H3/a antibody;H3/b antibody;H3/c antibody;H3/d antibody;h3/f antibody;H3/h antibody;H3/i antibody;H3/j antibody;H3/k antibody;H3/l antibody;H31_HUMAN antibody;H3F1K antibody;H3F3 antibody;H3FA antibody;H3FB antibody;H3FC antibody;H3FD antibody;H3FF antibody;H3FH antibody;H3FI antibody;H3FJ antibody;H3FK antibody;H3FL antibody;HIST1H3A antibody;HIST1H3B antibody;HIST1H3C antibody;HIST1H3D antibody
Pubmed ID(s): 26391951, 26436920, 24427299, 24829459, 24732798, 22707199, 22466965, 22223663, 22009739, 21097519, 21316602, 21695246, 18579779, 17968314, 16373353, 32376680, 31413827
Entrez Gene ID(s): 100364501, 100365669, 291159, 679994, 680511, 682330, 64009
SwissProt ID(s): Q6LED0, P68432, P02299

Citations

Citation Count: 17
Citations:

Ma P et al. RNF220 is required for cerebellum development and regulates medulloblastoma progression through epigenetic modulation of Shh signaling. Development 147:N/A (2020).
View Source


Nevoral J et al. Epigenetic and non-epigenetic mode of SIRT1 action during oocyte meiosis progression. J Anim Sci Biotechnol 10:67 (2019).
View Source


Protocols

Immunofluorescence protocol - Formaldehyde fixation
  1. Collect cells from T.c.unit and remove media from petri dish using suction.
  2. Wash with 1x PBS and remove.
  3. Incubate cells in pre-warm (37°C) Para-Formaldehyde for 12 minutes at room temperature on an orbital shaker.
  4. Remove PFA and incubate in 0.5% Triton X-IOO in 1x PBS for 5 minutes at room temperature.
  5. Prepare blocking reagent, this is also the antibody diluent.
  6. Wash cells 2x with 1x PBS at room temperature, for 4 minutes/wash on an orbital shaker.
  7. Block with 1 % NCS and 1x PBS for 30 minutes at room temperature.
  8. Prepare primary antibodies (50?l/coverslip) and moist staining chambers.
  9. Wash cells 2x with lx PBS at room temperature and air dry briefly.
  10. Incubate with primary antibody for 1 hr at room temperature in the dark in staining chambers. During this time prepare the secondary antibody.
  11. Wash cells 5x with 1x PBS (5 beaker changes/5 counts in each beaker)
  12. Incubate with secondary antibody for 1 hour at room temperature in the dark in staining chambers.
  13. Wash cells 5x with 1x PBS.
  14. Mount in Dapi.

Solutions (prepare fresh the same day of staining).

  • * 1x Phosphate buffered saline.
  • * Blocking reagent: 1% NCS in 1x PBS (use fresh l0x PBS).
  • * Fixation solution: 3.5% Para formaldehyde.

1.75g PFA in 20 ml d.H20 plus 5 drops 1M NaOH. Stir on a hot plate at 50-60°C until dissolved. Add 4 drops IN HCI and check pH indicator strip. PH should be 7.4. Complete volume with d.H20 to 25ml and add 25ml 2xPBS. Check pH before adding to cover slips.

Immunofluorescence protocol - Methanol/acetone fixation
  1. Collect cells from T.C.unit and remove media from petri dish using suction.
  2. Wash with 1x PBS and remove.
  3. Fix cells with cold methanol: acetone 1: 1 for 10 minutes on ice.
  4. Prepare blocking reagent, this is also the diluent for the antibodies.
  5. Remove fixative and wash cells 3x with Ix PBS at RT, for 4 minutes/wash on orbital shaker.
  6. Block with 1% NCS and Ix PBS for 30 minutes at RT.
  7. Prepare primary antibodies (50?l/coverslip) and moist staining chambers.
  8. Wash cells 2x with 1 x PBS at RT and air dry for approximately 7 minutes.
  9. Incubate with primary antibody for 1 hr at RT in the dark in staining chambers. During this time prepare secondary antibody.
  10. Wash cells 5x with 1x PBS (5 beaker changes/5 counts in each beaker)
  11. Incubate with secondary antibody for 1 hr at R T in the dark in staining chambers.
  12. Wash cells 5x with 1x PBS.
  13. Mount in Dapi.

Solutions (prepare fresh the same day of staining)

  • * 1x Phosphate buffered saline.
  • * Blocking reagent: 1% NCS in 1x PBS (use fresh 10x PBS).
  • * Fixation solution: methanol:acetone 1: 1 ice cold.
Western Blotting Protocol
  1. Transfer gel to PDVF or nitrocellulose membrane
  2. Place membrane in plastic tray in blocking buffer for one hour with agitation
  3. Rinse in wash buffer
  4. Incubate in wash buffer plus primary antibody for one hour
  5. Wash 6 X 5 minutes with wash buffer
  6. Incubate in wash buffer plus secondary antibody for one hour
  7. Wash 6X 5 minutes with wash buffer
  8. Detect (e.g. ECL, Amersham according to manufacturers instructions)
Wash buffer

PBS + 0.1% Tween 20

Blocking buffer

Wash buffer + 5% dried milk powder

The concentration of antibodies used depends on each antibody, the amount of antigen and the detection method used. Generally, dilution is in the range of a few hundred times dilution to a few thousand times dilution, but usually has to be determined empirically.

FAQ's

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If items are ordered incorrectly by the customer, ImmuQuest will consider taking them back as long as they have been stored correctly and have not been opened or tampered with. Such orders may be subject to a 15% restocking charge on the items plus any shipping costs.

Requests for returns must have prior authorization from ImmuQuest, and must be made within 7 days of receipt of the items. Items must be returned in the same or equivalent packaging as originally dispatched, and by an equivalent method of delivery.

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We use federal Express for UK & international shipments, andfor standard shipments the cost is £30 UK & £55 international. If the customer uses their account, we charge a £20 handling fee (UK & international)

We are strongly committed to providing the best quality products, and the best service possible.

Should any product not perform as described in the product literature, it will be replaced or a full refund will be given. The customer must notify ImmuQuest within 30 days after the goods have been received to request a replacement, forwarding complete test data as requested by ImmuQuest.

If items are ordered incorrectly by the customer, ImmuQuest will consider taking them back as long as they have been stored correctly and have not been opened or tampered with. Such orders may be subject to a 15% restocking charge on the items plus any shipping costs.

Requests for returns must have prior authorization from ImmuQuest, and must be made within 7 days of receipt of the items. Items must be returned in the same or equivalent packaging as originally dispatched, and by an equivalent method of delivery.

Product Guarantee

We are strongly committed to providing the best quality products, and the best service possible. In order to do this we depend on your feedback.

Should any product not perform as described in the product literature, it will be replaced or a full refund will be given. The customer must notify ImmuQuest within 30 days after the goods have been received to request a replacement, forwarding complete test data as requested by ImmuQuest.

If items are ordered incorrectly by the customer, ImmuQuest will consider taking them back as long as they have been stored correctly and have not been opened or tampered with. Such orders may be subject to a 15% restocking charge on the items plus any shipping costs.

Requests for returns must have prior authorization from ImmuQuest, and must be made within 7 days of receipt of the items. Items must be returned in the same or equivalent packaging as originally dispatched, and by an equivalent method of delivery.

If you have any questions please contact us using the following details:

ImmuQuest Ltd
Springboard
Stokesley Business Park
24 Ellerbeck Way
Stokesley
TS9 5JZ
UK

Tel: 01642 713533
Fax: 01642 713988
International Tel: +44 1642 713533
International Fax: +44 1642 713988

When placing an order ImmuQuest require a purchase order number, plus name and contact details of the purchaser, and the person who will be using the product. ImmuQuest will also need a VAT number for customers in the European Union.UK customers that are VAT exempt need to fax an exemption certificate.

Orders can be placed either by our website, via email or by mail. Please see our contact us page for more details. All orders are subject to availability. Prices of products do not include shipping, VAT or import duties where these are applicable.

Price and other information provided are subject to change without notice. While every effort is made to keep information provided Up to date, ImmuQuest will not be liable if errors should occur in such information. Formal acceptance of an order will take place when the goods are dispatched. If prices should be changed between the time of receipt of an order and dispatch, ImmuQuest will contact you in advance.