Following our previous work (24), [beta]-elimination with a mild alkylamine base and concomitant Michael-type addition of 2-mercaptoethanol
as nucleophile was applied.
0% (v/v) 2-mercaptoethanol
to prevent protein oxidation after cell lysis.
5,8] The serum samples were further tested with 2-mercaptoethanol
(2-ME) to confirm acute brucellosis infection.
Fifty mg of each tissue in RNAlater were homogenized in RTL lysis buffer (Qiagen) containing 1% 2-mercaptoethanol
2011), and the frozen isolates were macerated in a buffer lysis (beta 2-mercaptoethanol
, 2% SDS), 10% CTAB and 5N NaCl, followed by phenol extraction and the total DNA was resuspended in sterile TE.
3) Protease inhibitors stops proteolytic cleavage of antibody while reducing agents like dithiothreitol (DTT) and 2-mercaptoethanol
(2-ME) also prevents oxidation in the conjugate.
The digested tissue was removed to a 6-cm dish flooded with essential culture medium (alpha-minimum essential medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 [micro]g/mL Streptomycin, 2 mM L-glutamine, 5 x 10[sup]−5 M 2-mercaptoethanol
and 10[sup]−4 M L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate).
The cells was lysed with sampling buffer (2% SDS, 5% 2-mercaptoethanol
, 10% glycerol, and 0.
Sample buffer, with 5% 2-mercaptoethanol
, was added to 40 mg protein per lane and boiled for 2 min.
HEPES, penicillin, streptomycin, sodium bicarbonate, 2-mercaptoethanol
(2-ME), bovine serum albumin (BSA), histopaque, fetal calf serum (FCS), RPMI 1640 medium, M 199 medium were purchased from Sigma-Aldrich.
1% of 2-mercaptoethanol
using a sterile micropestle in 1.
19) On the other hand, on the slab SDS-PAGE in the absence of 2-mercaptoethanol
the purified enzyme showed two spots, 106 and 95kDa, both of which showed an identical amino acid composition and peptide mapping.