Sample buffer

Standardization—External standards, standard additions, and internal standards are a common feature of many quantitative analyses. Suggested experiments using these standardization methods are found in later chapters. A good project experiment for introducing external standardization, standard additions, and the importance of the sample s matrix is to explore the effect of pH on the quantitative analysis of an acid-base indicator. Using bromothymol blue as an example, external standards can be prepared in a pH 9 buffer and used to analyze samples buffered to different pHs in the range of 6-10. Results can be compared with those obtained using a standard addition. 

Why is the sample buffered to a pH of 10 What problems might be expected at higher or lower pHs ... 

Figure 2. SDS-PAGE of culture supernatants. Freeze-dried samples were resuspended in distilled water, mixed with an equal volume of sampling buffer and heated to 100°C for 5 min. lOpl aliquots were applied to the gel. The right lane contains standards of Mr 14-66 kDa. Lanes 2, 3, 4 and 1 are increasing dilutions of the supernatant respectively.

Figure 3. SDS-PAGE and in situ pectinase activity on pectin and polygalacturonic acid-agarose overlays of culture filtrates of Aspergillus niger N-402 (upper panel) and Aspergillus FP-180 (lower panel) at 2.5, 3.5, 5.5 and 6.5 pHi (Lanes a, b, c, and d, respectively). Electrophoresis on 10% acrylamide slab gel (14 X 8 cm) in the presence of SDS was according to Laemmli (6), run at 30 mA constant current for 2 hours. Crude cell-free samples were concentrated by lyophilization, dialyzed, boiled with sample buffer by 60 sec. and applied to each well. Polyacrylamide gel and overlays were incubated overnight with 0.17 acetate buffer at room temperature.

Centrifuge sample at maximum speed for 15 min at 4°. Remove supernatant and wash with ice-cold ethanol. Remove ethanol, air dry sample, and resuspend in sample buffer. 

Sample preparation 27 mg urea, 20 pi sample, 7 pi 7x7 sample buffer... 

Purification of eIF4Efrom cell lysates for IEF eIF4E and associated proteins are isolated from cell extract by affinity chromatography m7GTP-Sepharose (as described later) and the beads were washed twice with 1 ml of lysis buffer. 18 p of m7GTP (100 pM) is added to the beads and incubated for 15 min at 4°. After centrifugation at 7000 rpm for 30 s, the supernatant is mixed with 7x sample buffer and urea (see previously), and loaded onto prefocused IEF gel. 

Preparation of eIF2from cell lysates for IEF 60 /(I of fast-flow Sepharose S (prewashed with lysis buffer) and 500 pg of cell lysate are mixed for 2 h at 4°. After centrifugation, the supernatant is removed and the beads are washed twice with lysis buffer containing 200 mM KC1. The eIF2 should be eluted with 50 pi of lysis buffer containing 400 mMKCl. 20 pi ofeluate is then mixed with 7 X sample buffer and urea (see previously), and loaded onto a prefocused IEF gel. 

The immune complexes (beads) are washed 3 times with lysis buffer, and then 20 /il of 1 x SDS-PAGE sample buffer are added and the samples boiled for 5 min. The supernatant is subjected to SDS-PAGE, followed by western blotting. 

Reactions are allowed to proceed for 25 to 30 min at 30° with constant shaking at 900 rpm and then stopped by adding 8 tl of 5 x SDS-PAGE sample buffer, followed by boiling for 5 min. Supernatants are applied to 10% SDS-PAGE, followed by autoradiography or phosphorimaging. 

Analysis of the mRNA quality by PAGE/urea It is good practice to check the quality of the transcription product at various stages of the preparation. For this purpose, aliquots (e.g., 5 pi) of the reaction mix are taken at the beginning and end of the transcription reaction as well as after each step of the purification and mixed with an equal volume of electrophoresis sample buffer. After incubation at 65° for 5 min, the samples are loaded on a 6 to 8% acrylamide-7-8 M urea gel. 

SDS Sample Buffer for Running Electrophoresis Size Separations Under Reducing Conditions... 

Measure the absorbance of the solutions at 335 nm. Determination of the number of amines present in a particular sample may be done by comparison to a standard curve generated by use of an amine-containing compound (i.e., an amino acid) dissolved at a series of known concentrations in the bicarbonate sample buffer and assayed under identical conditions. 

Rinse the plated reaction vessel once with sample buffer to remove any loose particles of Iodogen that may not be strongly adhered to the surface of the glass. 

Wash cells and solubilize pellet in SDS-PAGE sample buffer. 

Quench the cleavage reaction by the addition of an equal volume of SDS electrophoresis sample buffer containing up to 40 percent glycerol. The SDS will denature the protein interaction and glycerol acts as a free radical scavenger, thus effectively quenching the reaction. 

C.A. Mauracher, L.A. Mitchell, and AJ. Tingle, Reduction of rubella ELISA background using heat denatured sample buffer. J. Immunol. Methods 145, 251—254 (1991). 

After mixing the sample, buffer, and internal standard, the mixture should be heated at 95 to 100°C for 10 to 12 min to complex proteins with SDS. For new proteins, it is advisable to prepare several vials of the same solution and heat them for various lengths of time in order to check for heat degradation artifacts. It is our experience that heating in a water bath is necessary use of a contact heating block is not always effective and may result in reduced separation efficiency. 

Sample dissolved in buffer that is 10 times more dilute than run buffer. If sample buffer cannot be diluted, increase run buffer ionic strength, however, not beyond point whereby excessive heat or current results (>100—200 pA) Reduce column ID Dip capillary in water rinse prior to high voltage (HV) start... 

---