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Proteins interacting with B2M in mouse synaptosome fractions

ABSTRACT: pGEX-4T-1-B2M was constructed using the GST gene fusion system according to the manufacturer’s instructions. To produce glutathione S-transferase (GST) and GST-B2M fusion proteins, pGEX-4T-1 and pGEX-4T-1-B2M were individually transfected into BL21 cells. Protein expression was induced by ispropylb-D-1-thiogalactopyranoside (IPTG). GST-B2M and GST proteins were purified by binding to Glutathione Sepharose-4B beads. Then, beads were incubated with mouse synaptosome fractions from C57BL/6J mice at 4 °C for 12 h on a rotator, and then were washed with the washing buffer three times. Beads-bound complexes were eluted by boiling, and subjected to SDS-PAGE and silver staining.

ORGANISM(S): Mus Musculus

SUBMITTER: Xin Wang

PROVIDER: PXD039275 | iProX | Wed Jan 04 00:00:00 GMT 2023

REPOSITORIES: iProX

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Project description:Post-hybridization washing is an essential part of microarray experiments. Both, the quality of the experimental washing protocol and the adequate consideration of washing in intensity calibration ultimately affect the quality of the expression estimates extracted from the microarray intensities. We conducted experiments on GeneChip microarrays with altered protocols for washing, scanning and staining to study the probe-level intensity changes as a function of washing cycles. Particularly, three Affymetrix GeneChip HGU133plus2 arrays were hybridized and equilibrated for 16 hours in the hybridization oven. For one of the three arrays washing and staining was performed according to the manufacturer’s instructions. For another array the first scan was done immediately after low stringent wash and staining without intermitting stringent washing. Then, the array was stringently washed and scanned in alternating order three more times where each washing step consists of a definite number of washing cycles. The third array was low stringently washed followed by two stringent washing cycles and staining before the first scan. Subsequently it was analogously processed as array A. All three chips are repeatedly processed in a second series of alternating wash/scan-cycles which was performed using the same protocol for each chip as in the first series as described above. As in the first series the arrays were also stained a second time to compensate for any loss of bleached fluorescent dye. Analysis of the washing kinetics shows that the signal-to-noise ratio doubles roughly every ten stringent washing cycles. Washing can be characterized by time-dependent rate constants which reflect the heterogeneous character of target binding to microarray probes. We propose an empirical washing function which estimates the survival of probe bound targets. The washing function allows calibrating probe intensities for the effect of washing. On a relative scale, proper calibration for washing markedly increases expression measures especially in the limit of small and large values.

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Proteins interacting with B2M in mouse synaptosome fractions

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