Product Review: Sdpr Antibody (AAS99504C) in Human placental and myometrial tissue lysate using Western Blot

Product Page for Sdpr antibody-N-terminal region (AAS99504C)

Researcher: Hiten Mistry, Ania Czajka and Marta Hentschke Ribeiro, King’s College London
Application: Western blotting
Species+tissue/cell type: Human placental and myometrial tissue lysate
How many ug’s of tissue/cell lysate run on the gel:
1: 30 ug human placental tissue lysate
2: 30 ug human placental tissue lysate
3: 30 ug human placental tissue lysate
4: 30 ug human placental tissue lysate
5: 20 ug human myometrial tissue lysate
Primary antibody dilution: 1:500
Secondary antibody: Goat anti-rabbit HRP
Secondary antibody dilution: 1:10000

Questionnaire:

How do Antibody Verify’s reagents play a role in your experimental goals?

It is one of our main antibodies of interest.

How would you rate this antibody on a scale from 1-5 (5=best) and why?

4. It did worked for tissue lysates of interest and for our positive control.

Would you use this antibody in future experiments?

Yes

Have you used another antibody which has worked in your application?

No

Do you believe the information about the reagent on Antibody Verify’s website is correct?

Yes

If the antibody works, do you plan to use it in future experiments or to publish your data? Why or why not?

Yes

How did you store the antibody after re-suspension?

-20C

Sample Description (please include 1) species type, and 2) cell/tissue type, and 3) how much protein loaded for each lane of your gel):

1. Human, 2. Placental lysate 3. 20-50ug/lane

How many different experimental trials were conducted using the antibody sample?

2-3

How was this sample prepared?

100mg of sample was homogenised in RIPA buffer and treated with protease inhibitors cocktail (SIGMA).

Primary antibody dilution and incubation time:

1:500, overnight incubation

Secondary antibody used and dilution and incubation time:

1:10000, 2-3 hrs

What controls were used in your experiment (positive/negative)?

Positive: myometrial tissue lysate

Please include your detailed WB Procedure/Protocol here:

Equipment: XCell SureLock Mini-cell system with blotting module, gel loading, pipette, tips, mechanical rocker.

1. Loading buffer: Laemlli buffer (Protein loaded (u/5) required to run for each sample)

2. Running buffer (MES SDS RB 20X)

3.Marker (10ug of Novex 4-12% Bis-Tris Gel 1X Mes Running buffer)

4. Gel: 4-12% Bis-Tris gel

5. Electrophorosis Conditions: 1h-1h30min, 175V

6. Transfer Membrane used: Immobilion-P (Millipore) (PVDF)

7. Block in methanol X2 (30 sec/15 sec)

8. Blotting conditions: 1h 30 min, 30V

9. Primary antibody (1:500), overnight Incubation

10. Wash 3 times for 20 minutes at maximum agitation in TBS-Tween after antibodies incubation

11. ECL (3mL per membrane, equal quantities of reagents A and B) for 1 minute

12. Blot analysis: Chemiluminescense (ECL TM)

13. Visualization useing GelLogic 2200 Pro, Carestream Program, from 10sec -30 mindepending on antibody

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