HOW DID OUR ANTIBODY PERFORM?

CUSTOMER REVIEW: Phospho-eIF2α (S51) Polyclonal Antibody (STJ90253)

WHAT WAS USED?Primary Antibody:

Provider:

Dilution ratio:

Secondary Antibody:

Application:

Materials for validation:

STJ90253 Phospho-eIF2α (S51)

Polyclonal Antibody

St John's Laboratory

1:500

Goat Anti-Rabbit IgG Antibody, (H+L) HRP conjugate (Sigma)

Western Blot.

Murine intestinal longitudinal muscle myenteric plexus (LMMP)

WHAT WAS THE PROTOCOL?Treatment of materials: LMMP were homogenized using a polytron tissue grinder in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, 0.25% wt/vol sodium deoxycholate, 0.1% Nonidet P-40, 100 M NaVO4) with protease inhibitors (Sigma). Samples were treated with loading buffer for 5 min in water bath at 100°C. Gel electrophoresis: 10% polyacrylamide gel, constant voltage 160 V, 60 min.Transfer: 0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 100 V, 90 min.Blocking: 1X TBST with 5% bovine serum albumin for 30 min.Primary antibody probing: primary antibody was added to the solution of 1X TBST with 5% bovine serum albumin, 4˚C for overnight.Membrane wash: 1X TBST washes for 3 times.Secondary antibody probing: Secondary antibody was added to the solution of 1X TBST with 5% bovine serum albumin with 1:2000 dilution ratio, for 45 min.Membrane wash: 1X TBST washes for 3 times.Visualization: ECL visualization for 5 min. Images were captured using Hyper Film MP (GE Healthcare).

WHAT WAS THE PROTOCOL?

Gel electrophoresis information： 10% polyacrylamide gel, constant voltage 160 V, 60 min.Transfer information： 0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 100 V 90 min.

No. Antigen Loading amount

Primary antibody

Primary antibody dilution

ratio

Secondaryantibody

dilution ratioTarget band KD Visualization

time

1LMMP from

C57Bl6J young mouse 20 g

STJ90253 Phospho-

eIF2 (S51) 1:500 1:2000 ~30KD 10S

2LMMP from

C57Bl6J aged mouse

WHAT DID THE CUSTOMER THINK?

Testing results were provided by Ignazio Castagliuolo from University of Padova, Italy. He stated that “In LMMP isolated from both young and aged C57Bl6J mice, there were target bands detected by STJ90253 Phospho-eIF2 (S51)

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