Precision plus protein kaleidoscope prestained protein ladder

Ethanol, mEthanol, chloroform, glacial acetic acid, HNO₃, H₂O₂, Na₂HPO₄, NaH₂PO₄, AgNO₃, Na₂CO₃ and sodium dodecyl sulphate (SDS) were purchased from Merck, Bengaluru, India. Bovine serum albumin and chitosan powder were purchased from Sisco Research Laboratories, Chandigarh, India. Glutaraldehyde was purchased from TCI Chemicals, Chandigarh, India. A Spurr resin kit and OsO₄ were purchased from Electron Microscopy Sciences, Delhi, India. Uranyl acetate was purchased from LobaChemie, India. CuCl, selenourea, oleylamine, citric acid, sodium citrate monobasic, lead citrate, Laemmli buffer, ammonium persulfate, trizma base, tetramethylethylenediamine (TEMED), acrylamide, N,N′- methylenebis(acrylamide), 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), KOH, phenylmethylsulfonyl fluoride (PMSF) cocktail, KH₂PO₄, K₂HPO₄, glycerol, and formaldehyde were purchased from Sigma-Aldrich, Bengaluru, India. Bradford reagent, macerozyme R-10, 1X phosphate buffer saline (PBS), sodium thiosulfate, and ascorbate were purchased from HiMedia, Mumbai, India. Rehydration buffer, IPG strips of pH range 3–10, a ReadyPrep 2D starter kit equilibration buffer I and II, and a ReadyPrep 2D clean-up kit was purchased from Bio-Rad, Gurugram, India. A precision plus protein kaleidoscope prestained protein ladder was purchased from Bio-Rad.

Four to 12% surePAGE gels (genescript) were used with MOPS running buffer (for higher molecular weight proteins) or MES running buffer (for lower molecular weight proteins). Gels were subjected to 200 V. A molecular weight marker Precision Plus Protein Kaleidoscope Prestained Protein Ladder was used (Biorad). Proteins were transferred to PVDF membranes with the eBlot L1 system using eBlot L1 Transfer Stack supports (Genscript) and the resulting membranes were washed three times with TBS-T (Tris-buffered saline containing 0.1% Tween 20 (Merk)). Membranes were blocked with 5% milk (Carl-Roth) in TBS-T at room temperature (RT) for 1 hr. Membranes were incubated at 4 °C with corresponding primary antibodies overnight (O/N). Membranes were washed three times with TBS-T for 10 min and incubated with secondary antibodies in 5% milk in TBS-T at RT during 1 hr. Membranes were washed three times with TBS-T for 10 min.