Application
Western blot
Sample
Mouse Cell lysate - whole cell (NIH/3T3 cells @ passage 22 (P1 = ATCC stock))
Gel Running Conditions
Reduced Denaturing (NuPAGE 4-12% Bis-Tris gel, 1X NuPAGE MOPS SDS Running Buffer (constant voltage, 100V, for 30min then 200V, for 50min))
Loading amount
50 µg
Treatment
~50,000 cells per well were seeded in a 24-well plate and reverse-transfected with pooled Stealth siRNA specifically targeting Lamp2a @ 30nM for ~46hrs and cell lysates were prepared using 1X RIPA buffer supplemented with protease inhibitors
Specification
NIH/3T3 cells @ passage 22 (P1 = ATCC stock)
Blocking step
Milk as blocking agent for 1 hour(s) and 15 minute(s) · Concentration: 5% · Temperature: 24°C
Other product details
Incubation time
14 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: with 1x TBST (0.1% Tween-20)
Dilution
1/500
Detection
Detection method
ECL+
Negative control
Cells transfected with Stealth RNAi control oligos (medium GC, 48%)
Exposure
1 minute(s) and 0 second(s)
Bands
Specific: ~90 kDa
Positive control
Cells transfected with Lamp2a-specific siRNA
Additional data
Additional Notes
Lysates were prepared by rinsing cells with 1ml EBSS per well and then incubated with 25ul 1x RIPA buffer (supplemented with protease inhibitors), on ice (~15min). Lysates were collected using a mini-scraper (United Biosystems, MCS-200) into a Protein Lo-Bind tube and then mixed with 15ul NuPAGE buffer (with 2.5X LDS Sample Buffer and 2.5X Reducing Agent). 3-4 1.4mm ceramic spheres (from a Lysing Matrix D 2ml tube) were added into each tube and processed on a bead beater for 30sec in a cold room, and then collected by centrifuged briefly @ 10,000X g @ 4°C. Lysates were then incubated @ 70°C, with shaking @ 500 rpm, ~5min. Samples were centrifuged @ 10,000X g, 5min, @ 4°C. Left on ice. ~1/3 lysates (15ul note: not sure about the exact quantity, 50ug was just an estimate) were used for electrophoresis.
For the transfer part - gel was transferred (without pre-equilibrating in transfer buffer), using the Trans-Blot Turbo mini (0.2um, PVDF Bio-Rad, 1704156) unit and transferred under standard protocol (25V, 1.0A, 45min - extended time).
The PVDF membrane was cut right underneath the 62-kDa marker (SeeBlue Plus2 Pre-stained Protein Standard, Invitrogen) and each piece was incubated with the designated primary antibody prep. All the other primary antibodies were diluted in 5% non-fat milk prepared in 1x TBST.
For ECL+ part - ~10min incubation with the reagent, exposed on ChemiDoc (15sec/5sec/60sec/300sec/120sec). The picture shown here refers to 60-sec exposure (signals of Gapdh and actin were saturated).
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
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投稿 Mar 04 2020