SDS-PAGE sample buffer

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. 

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

Add 50 pil of Elution Buffer to each well, mix on the MTP shaker (or vortex) for 1 min, place on the 96-well magnet for 1 min, and transfer the supernatant (eluate) to a fresh MTP for analysis on SDS-PAGE. SDS-PAGE sample buffer may be used Instead of elution buffer If you require a more concentrated sample for analysis. 

Sample buffer (non-reducing conditions). Mix equal volumes of water R and concentrated SDS-PAGE sample buffer R. 

Adding SDS to a final concentration of 0.05% and then dialyzing versus 1 mM NH4HCO3, 0.05% SDS followed by lyophilization in a Speedvac and reconstitution in SDS PAGE sample buffer. 

Figure 1. Apparent heterogeneity of humanized antibody hAB-1. Ten micrograms of each antibody were mixed with non-reducing or reducing SDS-PAGE sample buffer and boiled for ten minutes prior to electrophoresis. The NOVEX molecular weight markers (MW) are identified by their mass values. Samples Lanes 1,5 pMAB, lanes 2,6 hAB-2 and lanes 3,7 hAB-1. HC and LC correspond to heavy and light chains of antibodies, respectively.

Figure 2. Influence of sample preparation time on apparent heterogeneity. pMAB and hAB-1 were mixed with non-reducing SDS-PAGE sample buffer and boiled for various times prior to electrophoresis. Boiling times Lanes 1,2 1 minute, lanes 3,4 5 minutes and lanes 5,6 10 minutes. Samples pMAB (lanes 1,3 and 5), hAB-1 (lanes 2,4 and 6).

Figures. Influence of temperature on apparent heterogeneity. hAB-1 was incubated in non-reducing SDS-PAGE sample buffer for ten minutes at various temperatures prior to electrophoresis. Incubation temperatures Lane 1 5°C, lane 2 21°C, lane 3 37°C, lane 4 60 C and lane 5 boiling.

Figures. Role of a cysteine residue in autoreduction of hAB-1. hAB-1 (50 (ag) was incubated with either NEM (4 jtg) or lAA (200 Rg) in PBS at room temperature for 10 or 30 minutes. At the end of the incubation period, treated and untreated samples (10 pg) were boiled in non-reducing SDS-PAGE sample buffer and electrophoresed. Samples Lanes 1,4 untreated, lanes 2,5 treated with NEM and lanes 3,6 treated with lAA.

Remove a 0.5-mL sample of the culture for SDS-PAGE. Centrifuge for 1 min in a microcentrifuge. Discard the supernatant and resuspend the cell pellet in 75 pL of SDS-PAGE sample buffer. Store at -20° C. 

Resuspend protein A sepharose pellet in 100 pL IX SDS-PAGE sample buffer. Mix well but do not heat. Shake at room temperature for 15 min. 

Add the appropriate volume of 4x SDS-PAGE sample buffer (to get lx final concentration of the sample buffer) and boil the samples at 95 °C for 5 min. 

On ice, add 4 vol of 1% Triton X-100 and 1 mM PMSF, mix, then add an equal volume of twofold concentrated SDS-PAGE sample buffer. The sample can now be analyzed immediately or stored at — 20 C. 

Remove the supernatant and resuspend the membrane pellet in 1% Triton X-100, 1 mM PMSF, then add SDS-PAGE sample buffer before electrophoresis. 

The bound materials are eluted by boiling the pellet in an SDS-PAGE sample buffer and resolved on a 15% SDS-polyacrylamide gel for... 

Add 0.5 ml modified 2 X SDS-PAGE sample buffer and leave the cells at room temperature for 5 min with occasional swirling to ensure efficient extraction of proteins. 

To elute the immunoprecipitates for analysis by SDS-PAGE, add an equal volume of 2 X SDS-PAGE sample buffer, mix well, and heat at 100 C for 2 min. Pellet the beads by centrifugation at 13 000 g, and remove the supernatant. 

Reaction termination. The reactions are terminated by the addition of SDS-PAGE sample buffer (10 mM Tris-HCl, pH 6.8, 1 mM EDTA, 1% DTT, 2.5-5% SDS, 0 01% bromphenol blue, final) followed immediately by boiling. Prepare a 4X stock of SDS-PAGE sample buffer and store in aliquots at -20°C. Quickly boiling the samples is essential to minimize artifactual proteolysis owing to bindmg-protein denaturation... 

To two microtubes per reaction add 3 pL of 4X SDS-PAGE sample buffer. Keep the tubes on ice until use... 

Add 1/10 vol of protease stock and mix by gently flicking the tube. Quickly remove a 9-pL amount for the time-zero sample, add to SDS-PAGE sample buffer, immediately boil for 3 mm, and keep the boiled samples on ice... 

Incubate the remainder of the reaction at 37°C for 60 mm. After the incubation, briefly centrifuge the tube, mix, recentrifuge. This minimizes concentration of the protein owing to evaporation. Remove 9 pL and immediately boil in SDS-PAGE sample buffer for 5 mm During this, reboil the time-zero samples for 2 min... 

Heat water to boiling and prepare a senes of microtubes each with 3 pL SDS-PAGE sample buffer Store tubes on ice until needed... 

Take out 9-pL aliquots of the reaction mixture, add to SDS-PAGE sample buffer and boil as in Subheading 3.3. Recommended initial times are 0,5,10,15,30,45, and 60 min These may be vaned depending on the protease and substrate protein used,... 

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