globular protein size standards  (Fluka Chemical)

Journal: Alzheimer's & dementia : the journal of the Alzheimer's Association

Article Title: The aqueous phase of Alzheimer's disease brain contains assemblies built from ∼4 and ∼7 kDa Aβ species.

doi: 10.1016/j.jalz.2015.01.005

Figure Lengend Snippet: Fig. 5. Aqueous extracts of human brain contain up to four different sized Ab assemblies separable by size exclusion chromatography (SEC). Tris buffered saline (TBS) extract (1 ml) from one AD brain (AD33) was chromatographed on a Superdex 75 10/30 HR column (A). Half milliliter fractions were collected, a 0.25 ml aliquot was lyophilized and analyzed by western blotting (WB) using a combination of 2G3 and 21F12 (A) or a monoclonal antibody, 46-4, which does not recognize Ab (B). Bands specifically reactive with anti-Ab mAbs were detected in fxs (fractions) 1 and 2 (high molecular weight, High mwt), 3, and 4 (intermediate molecular weight, Int mwt) 17 and 18 (monomer). A darker exposure (A inset) is shown to highlight bands in fxs 17 and 18. Two milliliters of AD-TBS (AD28) was chromatographed on a Superdex 75 10/30 HR column linked in tandem with a Superdex 200 10/30 HR column (C). Fractions (1 ml) were lyophilized and analyzed by WB using 2G3/21F12. Because the fractions from a single chromatographic run could not be accommodated on a single gel, samples were electrophoresed on 2 (A) or 3 (C) gels alongside synthetic Ab1–42 standards (not shown) which were used to normalize the exposure of each blot. Species that migrated on sodium dodecyl sulfate polyacrylamide gel electrophoresis as monomer and dimer are indicated by arrows and molecular weight markers shown on the left, and fraction numbers are provided below each lane. Four peaks of Ab immunoreactivity were detected in the SEC fractions, and based on their elution relative to SEC standards are designated as (i) High mwt, (ii) Int mwt, (iii) w7 kDa Ab species and (iv) true monomer (monomer)— these species are highlighted with an underline. The elution of linear dextran standards is indicated by up-pointing arrows and labeled 5, 12, 25, 80, 150, 270 kDa, while the elution of globular standards, is indicated by down-pointing arrows labeled 1, 17, 44, 158, and 670 kDa. Fraction 0 indicates the peak fraction in which Blue dextran eluted. Nonspecific indicates bands detected when 46-4 was used.

Article Snippet: Globular protein size exclusion chromatography (SEC) standards were from Bio-Rad (Hercules, CA).

Techniques: Size-exclusion Chromatography, Saline, Western Blot, High Molecular Weight, Molecular Weight, Polyacrylamide Gel Electrophoresis, Labeling

Journal: Alzheimer's & dementia : the journal of the Alzheimer's Association

Article Title: The aqueous phase of Alzheimer's disease brain contains assemblies built from ∼4 and ∼7 kDa Aβ species.

doi: 10.1016/j.jalz.2015.01.005

Figure Lengend Snippet: Fig. 6. High and intermediate molecular weight oligomers are detected by enzyme-linked immunosorbent assay (ELISA) in aqueous extracts of Alzheimer’s disease (AD) brain fractionated on size exclusion chromatography (SEC) in physiological buffer. Tris-buffered saline extract (1 ml) from AD brain 30 was chromatographed on a Superdex 75 10/30 HR column eluted in 50 mM ammonium acetate (A and C) or artificial cerebrospinal fluid (aCSF) (B and C). Half milliliter fractions were collected and analyzed using the oligomer-specific ELISA (oELISA) (A and B) or the Abx-42 ELISA (C and D). High/interme- diate molecular weight oligomers were detected in fxs 1–4, whereas the majority of Abx-42 was found in fxs 14–18. Oligomer concentrations were calculated using an amyloid-b derived diffusible ligands (ADDL) standard with units of equivalent weight of Ab1–42 monomer (see [37]) and Abx-42 concentration were calculated based on the molar content of SEC-isolated Ab1–42 monomer. Fraction “0” was not examined by ELISA, and ND indicates that the concentration was not determined.

Article Snippet: Globular protein size exclusion chromatography (SEC) standards were from Bio-Rad (Hercules, CA).

Techniques: Molecular Weight, Enzyme-linked Immunosorbent Assay, Size-exclusion Chromatography, Saline, Derivative Assay, Concentration Assay, Isolation

Journal: Alzheimer's & dementia : the journal of the Alzheimer's Association

Article Title: The aqueous phase of Alzheimer's disease brain contains assemblies built from ∼4 and ∼7 kDa Aβ species.

doi: 10.1016/j.jalz.2015.01.005

Figure Lengend Snippet: Fig. 8. AD-TBS Ab treated with guanidium hydrochloride (GuHCl) elutes from size exclusion chromatography (SEC) and AF4 predominantly as monomer and dimer. A mixture of Ab1–42 monomer and DiYAb1–40 dimer was resuspended in 6M GuHCl, boiled for 10 minutes and chromato- graphed on a Superdex 75 10/30 HR column. One milliliter fractions were collected, lyophilized and analyzed by western blot (WB) using 2G3 and 21F12 (A). In a similar manner, AD-TBS (AD 21) was immunoprecipated with AW7 and the antigen-antibody complex eluted from Protein A sephar- ose beads by boiling in 6M GuHCl and chromatographed (B). One milliliter fractions were collected and analyzed by WB using 2G3 and 21F12 (B). The same samples as used in (A) and (B) were also analyzed using AF4. Samples were injected onto a 24.6-cm-long channel and 1 ml fractions were collected, lyophilized and analyzed by WB. Results for the synthetic Ab mixture and the AD-TBS extract are shown in (C) and (D), respectively. So- dium dodecyl sulfate polyacrylamide gel electrophoresis molecular weight markers are shown on the left, while elution of linear dextrans from SEC or AF4 are indicated by upward facing arrows. The elution of blue dextran is shown by an upward facing blue arrow for SEC (fraction [fx] 0), but for AF4 blue dextran eluted outside the range shown (fx 29), a horizontal arrow de- notes the later elution of blue dextran. No specific immunoreactive bands were detected above 10 kDa so blots were cropped accordingly. M refers to monomer and D to dimer.

Article Snippet: Globular protein size exclusion chromatography (SEC) standards were from Bio-Rad (Hercules, CA).

Techniques: Size-exclusion Chromatography, Western Blot, Injection, Polyacrylamide Gel Electrophoresis, Molecular Weight

Journal: Alzheimer's & dementia : the journal of the Alzheimer's Association

Article Title: The aqueous phase of Alzheimer's disease brain contains assemblies built from ∼4 and ∼7 kDa Aβ species.

doi: 10.1016/j.jalz.2015.01.005

Figure Lengend Snippet: Fig. 9. Size exclusion chromatography (SEC)-isolated amyloid beta (Ab) monomer and dimers have distinct primary structure compositions. Five milliliters of AD-TBS extract (AD43) was immunoprecipitated with AW7 (10 ! 0.5 ml), and the antigen-antibody complex eluted from the protein A sepharose beads by vortexing in 150 mM ammonium hydroxide (AmOH). The AmOH eluate was lyophilized, and the lyophilizate incubated in 6M guanidinium hydrochloride and then chromatographed on a Superdex 75 10/30 HR column. Half milliliter fractions were collected and 100 ml of each fraction was lyophilized and analyzed by WB using 2G3 and 21F12 (A). The remaining fractions were pooled in groups of 3 as indicated on the figure. These pools were lyophilized, dissolved in sample buffer and a sample of each pool (in duplicate) used for western blotting with 1G6 to detect N-terminally extended peptides and the same blot then serially reprobed with anti-Ab mAbs in the following order: 3D6, 6E10, and a combination of 2G3 and 21F12 (B). Molecular weight markers are shown on the left and the SEC origins of the samples are indicated below each lane. Exposures of the blots were adjusted so that the intensity of the synthetic Ab1–40 standards are comparable. Nonspecific indicates bands detected when 46-4 was used (not shown). Bands that migrate with molecular weights consistent for Ab monomer (M) and dimer (D) are indicated with arrows.

Article Snippet: Globular protein size exclusion chromatography (SEC) standards were from Bio-Rad (Hercules, CA).

Techniques: Size-exclusion Chromatography, Isolation, Immunoprecipitation, Incubation, Western Blot, Molecular Weight