Total proteins from long bones and cells were analyzed by western blotting. The bone powder and cells were lysed with RIPA buffer supplemented with 1% protease inhibitor cocktail (P0013B; Beyotime Biotechnology). To collect the supernatants, insoluble materials were removed by centrifuging at 13,300 rpm for 15 min. The protein amount was quantified with the bicinchoninic acid (BCA) protein assay (P0012; Beyotime Biotechnology) using different concentrations of bovine serum albumin (BSA) as a standard. Protein lysates were separated by electrophoresis in 10% polyacrylamide gels and were then transferred to polyvinylidene difluoride (PVDF) membranes. Subsequently, the membranes were blocked with 5% skimmed milk solution at room temperature and incubated at 4°C overnight with primary antibodies against BK (ab3586; Abcam, UK), β-catenin (51067-2-AP; Proteintech, USA), Runt-related transcription factor 2 (Runx2) (ab76956; Abcam), Col1a1 (A1352; Abclonal, USA), GSK3β (12456T; Cell Signaling Technology, USA), p-GSK3β (67558-1-Ig; Proteintech), USP7 (66514-1-Ig; Proteintech), and Axin1 (AF3287; R&D Systems, USA). The gray densities of the protein bands were all normalized by applying GAPDH density as an internal control.
Ab3586
Manufactured by Abcam
Sourced in United Kingdom
Ab3586 is a monoclonal antibody that detects the protein target X. It is intended for use in laboratory research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab76956, by Abcam (1 mentions)
A1352, by ABclonal (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Ab28508, by Abcam (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Alexa 546 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
3 protocols using ab3586
1
Quantitative Protein Analysis of Bone and Cell Lysates
2
Knockdown of TRPML-1 and BK Channels in U87MG Cells
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In U87MG cells, expression levels of TRPML1 and BK channels were knocked down using 1 μg/ml shRNA (Santa Cruz) against TRPML1 (Sc-44519) and BK channels (Sc-42511); control shRNA (Sc-108060) was used as a control. Jet prime reagent (2 μl) was used for transfection. Following 36 h incubation, 25 μg/ml puromycin (Invitrogen) was added to activate the shRNA promoter. After incubation for 2 to 3 days, cells were passaged to remove the dead and dying cells, and the remaining living cells were cultured for an additional 36 h prior to being taken for experimentation. Knockdown efficiency was confirmed by immunoblotting using antibodies against TRPML-1 (Abcam, Ab28508) and BK-channel (Abcam, Ab3586).
3
Multimodal Immunofluorescence Imaging of Neuronal Proteins
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Cells were fixed with 1% paraformaldehyde for 10 min followed by cold methanol (−20 °C) for 10 min. The cells were then washed with PBS, blocked with 5% goat serum, and incubated overnight at 4 °C with primary antibodies (1:100) against TRPML1 (Sc-398868, Santa Cruz), BK channels (Ab3586, Abcam) and LAMP1 (D2D11, Cell technology). After washing with PBS, cells were incubated with corresponding Alexa 488- or Alexa 546-conjugated secondary antibodies a(Invitrogen). Cells were examined by confocal microscopy (Zeiss LSM800). Controls for immunostaining specificity included staining neurons with primary antibodies without fluorescence-conjugated secondary antibodies (background controls), and staining neurons with only secondary antibodies; these controls helped eliminate auto-fluorescence in each channel and bleed-through (crossover) between channels.
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