Search

High-density lipoproteins (HDLs) are a heterogeneous mixture of lipoprotein particles with densities ranging from 1.063 to 1.021 g/ml. The various lipoprotein particles present in HDL have not yet been completely characterized, largely due to methodological difficulties. Asztalos and Schaefer have developed a powerful native-native 2D gel method to analyze HDL subpopulations with high resolution. In this technique, native HDL particles present in plasma are separated electrophoretically by charge in the first dimension and then by size in the second dimension. The native particles in the gel are then transferred onto a membrane using traditional Western blotting techniques. After probing the membrane with, for example, apoA-I antibodies, subpopulations of HDL particles containing apoA-I can be visualized. Traditionally, midi-sized native gels are poured manually in the laboratory and running, blotting, and probing the gels is a tricky and laborious procedure that involves the use of (125)I-labeled antibodies. Here we present a streamlined native-native 2D gel electrophoresis and blotting method using minigels. Traditional antibody incubation and chemiluminescent methods can be used for detection and use of (125)I is not required. This update of the Asztalos and Schaefer native-native 2D gel protocol renders the procedure more accessible to the nonspecialist.

Western analysis of apolipoproteins, lipoproteins, and proteins involved in lipoprotein metabolism can be challenging due to their size, hydrophobic nature, and, in some cases, low abundance. Here we describe a Western blotting method that has been used successfully for many proteins involved in lipoprotein metabolism, as well as intact LDL or HDL particles. Proteins or lipoprotein particles separated by gel electrophoresis are transferred to a PVDF membrane in a Hoefer TE22 transfer tank with Tris-Glycine-SDS-Methanol transfer buffer. The membrane is blocked with 3 % BSA/5 % milk to prevent nonspecific binding of antibody to the membrane and is then incubated with primary antibody that binds specifically to the protein of interest. After washing away unbound primary antibody, the membrane is then incubated with an HRP-labeled secondary antibody that binds primary antibody. After washing away unbound secondary antibody, the membrane is then incubated with a substrate for HRP, generating a chemiluminescent signal at the location of the protein of interest. The protein is visualized by exposing the membrane to an autoradiography film or an imaging device. Information on the use of several human antibodies, including apoA-I, A-II, apoB, apoC-II, apoC-III, apoD, apoL1, apoM, PON1, SAA, ABCA1, nitrotyrosine, and LCAT, is provided. This method can be used for Western blotting of virtually any protein as well as native lipoprotein particles.

Gene transcripts and transcript variants must be cloned to characterize gene function and regulation. However, obtaining full-length cDNAs with accurate sequences from the 5' end through to the 3' end can be challenging. Here we describe a reverse-transcriptase-based method for obtaining full-length cDNAs using the SMARTer ("Switching Mechanism At RNA Termini") RACE technology developed by Clontech. RNA is isolated from the tissue of interest and annealed to a primer (a modified oligo(dT) primer for polyA+ transcripts; random hexamers or a gene-specific primer for polyA- transcripts). A modified MMLV-reverse transcriptase uses the primer to initiate cDNA synthesis from RNA transcript(s) annealed to the primer and continues cDNA synthesis (reverse transcription) towards the 5' end of the transcript(s). Importantly, this reverse transcriptase possesses terminal transferase activity, so when it reaches the 5' end of a transcript it adds a 3-5 residue "tail" to the newly synthesized cDNA strand. Included in the reverse transcriptase reaction mix is an oligonucleotide containing a sequence tag as well as a terminal series of modified bases that anneal to the 3-5 residue tail on the newly synthesized cDNA. The reverse transcriptase proceeds from the end of the transcript onwards into the modified bases and the rest of the sequence-tagged oligo. The newly synthesized cDNA now has a sequence tag attached to it and can be used as a template for PCR, with one primer complementary to the sequence tag and the second primer specific to the gene of interest. The fragment can be cloned and sequenced or just sequenced directly. If high-quality, undegraded RNA is used, obtaining the true 5' end of a transcript is greatly enhanced. In combination with 3' RACE, full-length transcripts are easily cloned. This method provides sequence information on important regulatory regions, such as 5' and 3' UTRs and flanking regions, and is ideal for detecting transcript variants, including those with alternative transcriptional start sites, alternative splicing, and/or alternative polyadenylation.

Northern analysis detects and quantitates full-length RNA transcripts by hybridizing a labeled sequence-specific probe to RNA fractionated by size and blotted onto a membrane. The first step in Northern analysis is RNA extraction from the tissue(s) of interest, followed by RNA denaturation and electrophoresis to separate all RNA transcripts in the tissue by size. The size-fractionated RNA is then transferred (blotted) onto a membrane. The membrane is incubated with a labeled probe complementary to the sequence of interest. Stringent washing removes nonspecifically bound probe and leaves labeled probe bound specifically to the transcript(s) of interest on the membrane. Northern analysis provides information on gene transcript sizes and abundance and the presence of alternative splice variants or transcription initiation sites. Northern analysis can also confirm the presence or absence of specific exons within a given transcript. The method is medium throughput, as multiple samples can be analyzed side by side with relatively little effort. Finally, any potential PCR amplification artifacts are completely avoided using this methodology. Northern analysis offers a unique view of a gene's transcripts and provides novel insight into gene expression.