Ir conjugated secondary antibodies

Unless otherwise noted, all chemicals and reagents were purchased from Sigma Aldrich (St. Louis, MO). Details for all antibodies used in this work are listed in Table 1.
Briefly, polyclonal antibodies were generated to AnkG from amino acids TEDK to KKTH, to βIV Spectrin from amino acids ARRA to QESA, and to pan-Na_V channels from amino acids FNQQ to AFDI of the sodium channel Na_V1.6. Other antibodies used are anti-Caspr (Bhat et al., 2001 (link)), anti-NF186 (Thaxton et al., 2011 (link)), and anti-NFCT (Pillai et al., 2009 (link)). Commercial antibodies used included rabbit anti-myelin basic protein (Abcam; Cambridge, MA); mouse anti-α-tubulin (DSHB; Iowa City, IA); mouse anti-Caspr, anti-AnkR and anti-Kv1.2 (NeuroMab; Davis, CA), fluorescent secondary antibodies (Alexa Fluor; Life Technologies; Grand Island, NY), and infrared (IR) conjugated secondary antibodies (LI-COR; Lincoln, NE). All electron microscopy reagents were purchased from Electron Microscopy Sciences (Hatfield, PA).

Cells were lysed with RIPA buffer supplemented with protease inhibitor tablets and phosphatase inhibitor cocktail. Protein concentration was quantified using Pierce BCA protein assay kit following manufacturer’s protocol. 40 micrograms of protein were loaded into GenScript SurePage 4%-12% polyacrylamide gel, transferred to both nitrocellulose and PVDF membranes, blocked in 5% BSA or 5% milk in 1x TBST for one hour, before incubating the membranes with primary antibodies overnight at 4°C in 1% BSA solution. Membranes were washed with 1x TBST 3 three times before incubating the membranes in LI-COR IR-conjugated secondary antibodies (1:10,000-20,000) for 2 hours at room temperature. Membranes were washed three times with 1x TBST and imaged using the LI-COR Odyssey System. Membranes were adjusted and quantified with the LI-COR Image Studio Lite software (v5.2). The following antibodies were used for Western Blot Analysis: At 1:1000 Total AR (rabbit CST: #5153), Total SRC (rabbit CST: #2109), Total β-actin (mouse Santa Cruz: 4970S), ARpY534 (rabbit Invitrogen: #PA5-64643), SRCpY416 (rabbit CST: #2101), FKBP5 (rabbit CST: #8245), NKX3.1 (rabbit CST: #83700) cleaved PARP (mouse CST: #32563), total PARP (rabbit CST: #9532). At 1:500, ARpS81 (rabbit Sigma-Aldrich: #07-1375), AR-V7 (rabbit CST: #19672). Blots are results in triplicate.