Anti-ZC3H13 antibody (ab70802)

Thanks for the reply. In the data sheet that came along with anti-ZC3H13 antibody (ab70802), you have recommended a dilution of 1/2000 - 1/10000 for the Western blot. However, the image of the blot shown in the data sheet says that the antibody was used at a concentration of 0.04 microgram/ml, which seems to be a dilution of 1/25000. I would like to know the concentration of anti-ZC3H13 and the secondary antibody used in your blots.

ANSWER:

Thank you for your reply.

The concentration of ab70802 is 1 mg/ml. Yes, the recommended antibody dilution is 1/2000 - 1/10000. But the antibody can be used at other dilutions as well, dependenton the samples being studied.

As secondary antibody any HRP-conjugated anti-rabbit secondary antibody can be used, such as e.g.ab16284 (http://www.abcam.com/Rabbit-IgG-secondary-antibody-ab16284.html) or ab97051 (http://www.abcam.com/Goat-polyclonal-Secondary-Antibody-to-Rabbit-IgG-H-L-HRP-ab97051.html).

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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send Western blot protocol

ANSWER:

Thank you for contacting us.

The following WB protocol has been used with this antibody, togetherwitha 3-8% SDS Precast gel and HeLa whole cell lysate.

Western Blot Protocol

Reagents Needed:

20X Running Buffer


Tricine (free base) 71.7 g
Tris (free base) 72.6 g
SDS 10.0 g
Sodium Bisulfite 2.5 g



Adjust to 500 ml with ultra pure water.

Store at 4°C. For 1X Running Buffer, add 10 ml of 20X Running Buffer to 190 ml of distilled water.

10X Transfer Buffer


Tris (free base) 15.2 g
Glycine 72.1 g
SDS 5.0 g



Ultra pure water to 500 ml

Store at 4°C.

1X Transfer Buffer


10X Transfer Buffer 50 ml
Methanol 100 ml
Distilled water 350 ml



Make fresh for each use.

5% non-fat dry milk in TBST


Carnation non-fat dry milk 50 g
TBST 1 liter



TBST (Tris Buffered Saline with Tween 20, pH8.0)


Tris 6.1 g
NaC l 8.68 g
Tween-20 500 mcl



Adjust the volume to 1 liter with distilled water. Adjust pH to 8.0 with HCL.

Store at 4-25°C.

Electrophoresis in SDS-Polyacrylamide Gel and Transfer to Nitrocellulose:

Molecular weight markers:ab116027, ab116028, ab115832, ab119210.
Nitrocellulose membranes.

Cut open the package that contains the gel cassette and drain away the buffer.
Rinse the wells with distilled water.
Rinse the wells with fresh 1x running buffer.
Place the gels on the buffer core so that the shorter plates face inward. If only using one gel, use a buffer dam to seal the other side.
Fill the inner core with fresh 1X running buffer. If there are no leaks, fill the outer core with running buffer. Load samples.
Run the gels at 150V until the dye front reaches the bottom of the gel (approximately 60 minutes).
Soak nitrocellulose membrane and blotting paper in 1X transfer buffer for at least 5 minutes prior to opening gel cassette.
Open gel cassettes and place the gel on the nitrocellulose membrane sandwiched between two pieces of blotting paper.
Place in transfer apparatus and fill with fresh 1X transfer buffer.
Run transfer apparatus for 60-75 minutes on 35V.



Western Blotting:

Remove the membrane from the transfer apparatus and place in 20 ml of 5% non-fat dry milk in TBST for one hour, with gentle shaking.
Dilute the primary antibody in 15 ml of 5% non-fat dry milk in TBST. For best results, the optimal dilution of antibody should be empirically defined.
Incubate the membrane in diluted primary antibody for two hours to overnight with gentle shaking at room temperature.
Wash the membrane three times, 10 minutes each time in TBST.
Dilute the secondary HRP conjugated antibody in 15 ml of reconstituted 5% non-fat dry milk in TBST. For best results, the optimal concentration of the secondary HRP conjugated antibody should be empirically defined.
Incubate the membrane in diluted anti-Rabbit IgG-HRP Conjugate for 60 minutes.
Wash as directed in step 4.
Develop blots with substrate solution and place in plastic membrane protector.
Expose membrane to film or CCD camera.




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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