Question (59407) | Anti-Cleaved Caspase-3 antibody (ab2302)

WB Questionnaire


1) Abcam product code: ab2302

2) Abcam order reference number or product batch number: 1102605

3) Description of the problem: We have been testing an antibody anti-active Caspase 3 that was purchased from Abcam (ab2302). When we tested the positive control that you suggest (Camptothecin 2 µM treated Jurkat cells) we obtained a very weak signal using the antibody at the concentration 1:100. We already tried several protein concentrations as well as different denaturation temperatures and transfer times. The results were always as you can see in the picture or even worst. Hence, we would like to know if you have any suggestion.

4) Sample preparation:
Type of sample (whole cell lysates, fraction, recombinant protein…): whole cell lysate
Lysis buffer: RIPA
Protease inhibitors: protease inhibitor cocktail provided with RIPA
Phosphatase inhibitors: PMSF and NaOv provided with RIPA
Reducing agent: Tris-Glycine SDS sample buffer with mercaptoethanol
Boiling for ≥5 min? Yes
Protein loaded ug/lane or cells/lane: 30 µg
Positive control: Camptothecin 2 µM treated Jurkat cells
Negative control: Jurkat cells

5) Percentage of gel: 12.5%
Type of membrane: Nitrocellulose
Protein transfer verified: Yes
Blocking agent and concentration: 5% non-fat dry milk in TBST
Blocking time: 1 hour
Blocking temperature: room temperature

6) Primary antibody (If more than one was used, describe in “additional notes”): Anti-active Caspase 3 (ab2302)

Concentration or dilution: 1:100

Diluent buffer: 5% non-fat dry milk in TBST

Incubation time and temperature: Overnight at 4ºC



7) Secondary antibody: goat anti-rabbit Biorad

Species: Goat

Reacts against: Rabbit

Concentration or dilution: 1:3000

Diluent buffer: 5% non-fat dry milk in TBST

Incubation time: 1 hour

Incubation temperature: room temperature

Fluorochrome or enzyme conjugate: horseradish peroxidase



8) Washing after primary and secondary antibodies: yes

Buffer: TBST

Number of washes: 3



9) Detection method: chemiluminescence



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

Do you obtain the same results every time? Yes

What steps have you altered to try and optimize the use of this antibody? We altered several steps but the best results were obtained with the conditions presented in the questions.



Boiling for ≥5 min? Yes/No

Protein loaded ug/lane or cells/lane: 30 µg/50 µg

Percentage of gel: 10%/12.5%

Incubation time and temperature: Overnight at 4ºC and 1 hour at room temperature

Concentration or dilution of the caspase 3 antibody: 1:100, 1:200; 1:250, 1:500


The image attached correspond to the conditions presented in the questions above.