Anti-CASC5/KNL1 抗体 (ab70537)

Thank you for your recent enquiries.

I am pleased to provide you with a copy of the western blot protocol used to test this antibody. Please note as previously discussed that this would be a guideline protocol only, and may require some further optimization by different customers.

I hope this will be helpful to you. I look forward to hearing from you with further details.


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 4oC.
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

10X Transfer Buffer is available commercially. Usually stored at 4oC, but check manufacturers instructions.

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 4-25oC.


Electrophoresis in SDS-Polyacrylamide Gel and Transfer to Nitrocellulose:
Many of the reagents for these procedures are commercially available.

1.Cut open the package that contains the gel cassette and drain away the buffer.
2.Rinse the wells with distilled water.
3.Rinse the wells with fresh 1x running buffer.
4.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.
5.Fill the inner core with fresh 1X running buffer. If there are no leaks, fill the outer core with running buffer. Load samples.
6.Run the gels at 150V until the dye front reaches the bottom of the gel (approximately 60 minutes).
7.Soak nitrocellulose membrane and blotting paper in 1X transfer buffer for at least 5 minutes prior to opening gel cassette.
8.Open gel cassettes and place the gel on the nitrocellulose membrane sandwiched between two pieces of blotting paper.
9.Place in transfer apparatus and fill with fresh 1X transfer buffer.
10.Run transfer apparatus for 60-75 minutes on 35V.

Western Blotting:
1.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.
2.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.
3.Incubate the membrane in diluted primary antibody for two hours to overnight with gentle shaking at room temperature.
4.Wash the membrane three times, 10 minutes each time in TBST.
5.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.
6.Incubate the membrane in diluted anti-Rabbit IgG-HRP Conjugate
or anti-Goat IgG-HRP Conjugate for 60 minutes.
7.Wash as directed in step 4.
8.Develop blots with substrate solution and place in plastic membrane protector.
9.Expose membrane to film or CCD camera.

Thank you for your telephone call this morning. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

I would like to reassure you that this antibody is tested and covered by our 6 month guarantee forWB and human samples. In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

As discussed on the telephone, I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily.

I would appreciate if you could also provide an image, including molecular weight markers,which would help us to assess the results.

I am also contacting the originator of this antibody to request the western blot testing protocol.

Thank you for your time and cooperation.I look forward to receiving the completed questionaire.



Order Details
Antibody code:

Problem
Choose: Non-specific band Multiple bands No signal or weak signal High background

Lot number

Purchase order number
or preferably Abcam order number:



General Information
Antibody storage conditions (temperature/reconstitution etc)


Description of the problem (high background, wrong band size, more bands, no band etc.)


Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)


Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)


Amount of protein loaded


Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)


Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)


Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Detection method (ECL, ECLPlus etc.)


Positive and negative controls used (please specify)




Optimization attempts (problem solving)
How many times have you tried the Western?



Have you run a "No Primary" control?
Yes No

Do you obtain the same results every time?
Yes No
e.g. are the background bands always in the same place?


What steps have you altered?


Additional Notes:


Image:
We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to asess the results.