Question (66465) | Anti-Dnmt1 antibody [60B1220.1] - ChIP Grade (ab13537)

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Question

Thanks for your previous email. Just as a follow on
question what negative control would you recommend and for
Anti-Dnmt1 antibody [60B1220.1] is there a specific IHC
protocol(other that the generic protocol on your website)
that you follow as I couldn't find it on the data sheet.
Many thanks

Answer

Thank you for contacting us.

If there is no other protocol then our standard protocols suggested on the product data sheet, the general protocols should be sufficient. However, I contacted the laboratory to get the protocol that was used doing the testing of the antibody. The protocol used is as followed:

Immunohistochemistry protocol for Antibody detection in formalin fixed tissues for Antibody Detection in Formalin-fixed for Antibody Tissues

Tissue Preparation: Formalin fixation and embedding in paraffin wax

Tissue Sectioning: Make 4-μm sections and place on pre-cleaned and charged microscope slides.

Heat in a tissue-drying oven for 45 minutes at 60°C.

Deparaffinization: Wash dry slides in 3 changes of xylene – 5 minutes each @ RT.

Rehydration: Wash slides in 3 changes of 100% alcohol – 3 minutes each @ RT.

Wash slides in 2 changes of 95% alcohol – 3 minutes each @ RT.

Wash slides in 1 change of 80% alcohol – 3 minutes @ RT.

Rinse slides in gentle running distilled water – 5 minutes @ RT.

Antigen retrieval: Steam slides in 0.01 M sodium citrate buffer, pH 6.0 at 99-100°C - 20 minutes.

Remove from heat and let stand at room temperature in buffer - 20 minutes.

Rinse in 1X TBS with Tween (TBST) – 1 minute @ RT.

Immunostaining: Do not allow tissues to dry at any time during the staining procedure

Apply a universal protein block – 20 minutes @ RT.

Drain protein block from slides, apply diluted primary antibody – 45 minutes @ RT.

Rinse slides in 1X TBST - 1 minute @ RT.

Apply a biotinylated anti-rabbit IgG (H+L) secondary – 30 minutes @ RT.

Rinse slides in 1X TBST - 1 minute @ RT.

Apply alkaline phosphatase streptavidin – 30 minutes @ RT.

Rinse slides in 1X TBST - 1 minute @ RT.

Apply alkaline phosphatase chromogen substrate – 30 minutes @ RT.

Wash slides in distilled water – 1 minute @ RT.

Dehydrate: This method should only be used if the chromogen substrate is alcohol

insoluble (e.g. Vector Red, DAB)

Wash slides in 2 changes of 80% alcohol – 1 minute each @ RT.

Wash slides in 2 changes of 95% alcohol – 1 minute each @ RT.

Wash slides in 3 changes of 100% alcohol – 1 minute each @ RT.

Wash slides in 3 changes of xylene – 1 minute each @ RT.


Apply coverslip.
Please be aware that any protocol may need optimisation for each experiment carried out.

Regarding the question for a negative control, according to "The Human Protein Atlas" Dnmt1 seems not to be expressed in for example in CNS (brain) and Liver/Pancreas. You might want to look at the webpage http://www.proteinatlas.org/ENSG00000130816 and confirm the information by some literature search.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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