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Supplemental Material
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Supplementary Material, Fig. S1 a. Titration of the antisera for HDAC6 Antibody titres
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were derived by Indirect ELISA using different dilution of antisera and pre-immune sera
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against fixed concentration of peptide (1µg/well). Peptide-antibody binding was detected
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using HRP conjugated Swine anti-rabbit IgG and TMB/H2O2 as substrate and
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measuring Absorbance (OD) at 450nm. Data represents Mean ± SEM of triplicates for
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each dilution. The titres were observed to be 1:4000 at an OD of 1. An increase in the
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absorbance at increasing concentrations of the antipeptide antisera (post-imm) and
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baseline absorbance at all dilutions for pre-immune (pre-imm) sera was observed. b.
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Competitive ELISA to determine the specificity of the antipeptide antibody. Specificity of
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the antibody was determined by competitive ELISA by preincubating the antipeptide
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antibody (1:4000) with 0, 0.25, 0.5, 1, 2, 4 and 8g of either corresponding peptide
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(DPSVLYVSLYVSLHRYGGYMNEGELR) or unrelated peptide (VVDSEDLPLN) and
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then using the preincubated mixtures to probe the peptide immobilized on to microtitre
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plates. Preimmune sera preincubated with peptide served as a control. The antipeptide
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antibody preincubated with the peptide (immunogen) showed a decrease in absorbance
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with increasing peptide concentrations as against the similarly treated preimmune sera
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which showed baseline absorbance. Antipeptide antibody preincubated with unrelated
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peptide showed no change in the absorbance at increasing concentrations of the
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unrelated peptide. This data demonstrates the specificity of the antipeptide antibody. c.
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An absorption control experiment using blocking peptide was performed to confirm the
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specificity of anti-peptide antibody. Towards this, caudal sperm proteins resolved by
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SDS PAGE and transblotted on NC membrane were probed with either the antipeptide
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(HDAC6) antibody (Ab), or, antipeptide antibody pre-absorbed with blocking peptide
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(Ab+BP), or antipeptide antibody pre-absorbed with unrelated peptide (Ab+UR). The
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lane probed with anti-peptide antibody pre-absorbed with blocking peptide showed
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marked (~80%) decrease in the intensity of HDAC6 band at 130kDa as compared to the
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anti-peptide antibody which was pre-absorbed with unrelated peptide, or antipeptide
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antibody used without preabsorbtion. The negative control was probed with pre-immune
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serum. Beta-actin is used as loading control.