Question (36856) | Anti-Rad17 (phospho S645) antibody (ab3620)

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Question

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1) Abcam product code ab3620

2) Abcam order reference number or product batch number GR686-4

3) Description of the problem

No signal

4) Sample preparation:

Type of sample (whole cell lysates, fraction, recombinant protein…) whole cell lysates

Lysis buffer RIPA buffer

Protease inhibitors: Protease inhibitor cocktail

Phosphatase inhibitors Phosphatase inhibitor cocktail

Reducing agent SDS

Boiling for ≥5 min? yes, 10 min

Protein loaded ug/lane or cells/lane 51 ug

Positive control n/a

Negative control n/a

5) Percentage of gel 4-20% gradient gel

Type of membrane PVDF

Protein transfer verified ponceau reagent

Blocking agent and concentration 5% milk in TBST

Blocking time 1 hour

Blocking temperature room temperature

6) Primary antibody (If more than one was used, describe in “additional notes”) :

Concentration or dilution 1/500

Diluent buffer 5% milk in TBST

Incubation time overnight

Incubation temperature: 4 degree


7) Secondary antibody:

Species:Donkey

Reacts against: rabbit

Concentration or dilution 1/5000

Diluent buffer 5% milk in TBST

Incubation time 1 hour

Incubation temperature: room temperature

Fluorochrome or enzyme conjugate:HRP

8) Washing after primary and secondary antibodies:

Buffer TBST

Number of washes 3 times, 5 minutes each

9) Detection method Chemiluminascence

10) How many times have you run this staining? twice

Do you obtain the same results every time? yes

What steps have you altered to try and optimize the use of this antibody? no

Answer

Thank you for your enquiry regarding ab3620 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.

The protocol looks fine to me however I would like to make few comments/suggestions that might help to improve your results and that you could consider trying:

ab3620 is a phospho specific antibody. We used 10Gy IR treated Hela cells to test the specificity of this antibody. I would recommend treating your sample cells there by inducing the phosphorylatuion of amino acid chemically or by radiations.

Milk is not recommended as blocking reagent with phospho specific antibodies. Please use BSA.

Please try both of these suggestions and let us know the results; we will be happy to help further.

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